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Mitochondrial dysfunction in high-fat diet-induced nonalcoholic fatty liver disease: The alleviating effect and its mechanism of Polygonatum kingianum
Biomedicine & Pharmacotherapy 117 (2019) 109083
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Mitochondrial dysfunction in high-fat diet-induced nonalcoholic fatty liver disease: The alleviating effect and its mechanism of Polygonatum kingianum
T
Xing-Xin Yanga,b,1, Xi Wanga,b,1, Ting-Ting Shic, Jin-Cai Donga,b, Feng-Jiao Lia,b, Lin-Xi Zenga,b, ⁎ Min Yanga,b, Wen Gua,b, Jing-Ping Lia,b, Jie Yua,b, a
College of Pharmaceutical Science, Yunnan University of Chinese Medicine, 1076 Yuhua Road, Kunming, 650500, China Kunming Key Laboratory for Metabolic Diseases Prevention and Treatment by Chinese Medicine, 1076 Yuhua Road, Kunming, 650500, China c Department of Pharmaceutical Preparation, The Xixi Hospital of Hangzhou Affiliated to Zhejiang University of Traditional Chinese medicine, Hangzhou, 310023, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: High-fat diet Mechanism Mitochondria Nonalcoholic Fatty liver Polygonatum kingianum Protective effect
Background: Mitochondrial dysfunction is an important mechanism of non-alcoholic fatty liver disease (NAFLD). Developing mitochondrial regulators/nutrients from natural products to remedy mitochondrial dysfunction represent attractive strategies for NAFLD therapy. In China, Polygonatum kingianum (PK) has been used as a herb and food nutrient for centuries. So far, studies in which the effects of PK on NAFLD are evaluated are lacking. Our study aims at identifying the effects and mechanism of action of PK on NAFLD based on mitochondrial regulation. Methods: A NAFLD rat model was induced by a high-fat diet (HFD) and rats were intragastrically given PK (1, 2 and 4 g/kg) for 14 weeks. Changes in body weight, food intake, histological parameters, organ indexes, biochemical parameters and mitochondrial indicators involved in oxidative stress, energy metabolism, fatty acid metabolism, and apoptosis were investigated. Results: PK significantly inhibited the HFD-induced increase of alanine transaminase, aspartate transaminase, total cholesterol (TC), and low density lipoprotein cholesterol in serum, and TC and triglyceride in the liver. In addition, PK reduced high density lipoprotein cholesterol and liver enlargement without affecting food intake. PK also remarkably inhibited the HFD-induced increase of malondialdehyde and the reduction of superoxide dismutase, glutathione peroxidase, ATP synthase, and complex I and II, in mitochondria. Moreover, mRNA expression of carnitine palmitoyl transferase-1 and uncoupling protein-2 was significantly up-regulated and down-regulated after PK treatment, respectively. Finally, PK notably inhibited the HFD-induced increase of caspase 9, caspase 3 and Bax expression in hepatocytes, and the decrease of expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. Conclusion: PK alleviated HFD-induced NAFLD by promoting mitochondrial functions. Thus, PK may be useful mitochondrial regulators/nutrients to remedy mitochondrial dysfunction and alleviate NAFLD.
1. Introduction Non-alcoholic fatty liver disease (NAFLD), which is characterized as an abnormal accumulation of triglycerides in hepatocytes, isnow considered the most prevalent type of chronic liver disease worldwide with
an incidence that is even higher with respect to liver damage caused by hepatitis C virus or alcohol abuse [1]. NAFLD is a histological disease, which may progress from simple steatosis to nonalcoholic steatohepatitis, liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma [2,3].
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; ATPase, ATP synthase; BCA, bicinchoninic acid; CPT-1, carnitine palmitoyl transferase-1; GSH-PX, glutathione peroxidase; HDL-C, high density lipoprotein cholesterol; HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; HRP, horseradishperoxidase; LDL-C, low density lipoprotein cholesterol; LPK, low-dose of Polygonatum kingianum; MDA, malondialdehyde; MPK, middle-dose of Polygonatum kingianum; NAFLD, non-alcoholic fatty liver disease; NC, normal control; PK, polygonatum kingianum; RES, resveratrol; ROS, reactive oxygen species; RT-PCR, real-time quantitative reverse transcription polymerase chain reaction; SD, sprague–Dawley; SOD, superoxide dismutase; TC, total cholesterol; TG, triglyceride; UCP-2, uncoupling protein-2 ⁎ Corresponding author at: College of Pharmaceutical Science, Yunnan University of Chinese Medicine, 1076 Yuhua Road, Kunming, 650500, China. E-mail address: [email protected] (J. Yu). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109083 Received 16 February 2019; Received in revised form 3 June 2019; Accepted 3 June 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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(AST), triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and ATP synthase (ATPase) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). In addition, kits to determine respiratory chain complex I and II were provided by Genmed Scientifics Inc. (Arlington, MA, USA). Bcl-2 mouse monoclonal antibody was purchased from Shanghai Jiwei Biological Technology Co., Ltd. (Shanghai, China). Cytchrome c mouse monoclonal antibody, β-actin mouse monoclonal antibody, horseradish-peroxidase (HRP) AffiniPure Goat Anti-Mouse IgG (H + L) antibody and HRP AffiniPure Goat Anti-Rabbit IgG (H + L) antibody were purchased from the Proteintech Group (Chicago, IL, USA). Caspase 3 rabbit monoclonal antibody, caspase 9 rabbit monoclonal antibody, and Bax rabbit monoclonal antibody were purchased from Abcam (Cambridge, UK). Primers were designed by Servicebio (Wuhan, China). TRIzol®Reagent was purchased from Ambion (Carlsbad, CA, USA). RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA), and FastStart Universal SYBR Green Master (Rox) was purchased from Roche (Basel, Switzerland). Highly Efficient RIPA Tissue/Cell Lysate was provided by Solarbio Science & Technology Co., Ltd. (Beijing, China). Immobilon™ Western Chemiluminescence HRP Substrate was obtained from Millipore Corporation (Bedford, MA, USA). All other reagents were of analytical grade or higher. The rhizome of PK (purchase date April 07, 2017) was purchased from Wenshan Shengnong Trueborn Medicinal Materials Cultivation Cooperation Society (Wenshan, China). Samples were authenticated by Professor Jie Yu, and a voucher specimen of PK (No. 8426) was deposited in the Key Laboratory of Preventing Metabolic Diseases of Traditional Chinese Medicine, Yunnan University of Chinese Medicine (Kunming, China).
To date, no approved drugs are available to treat patients with fatty liver disease and clinical management strategies mainly rely on modifications of diet, physical activity, and lifestyle, as well as on correcting hyperglycemia, insulin resistance, and hyperlipidemia, which are metabolic disturbances associated with NAFLD [3]. Therefore, it is of great value to develop treatment regimens and identify drugs/nutrients to alleviate NAFLD. Mitochondria are play a central role in complex processes, including the generation of energy and reactive oxygen species (ROS), maintaining calcium homeostasis, and adjusting apoptosis and lipid metabolism [4]. In an increasing number of studies, it has been demonstrated that mitochondrial dysfunction is an important mechanism of NAFLD and its aggravation and in which the following mechanisms may play a role [5–12]: 1) mitochondrial DNA damage; 2) disorders of energy metabolism; 3) oxidative stress and lipid peroxidation; 4) mitochondria-mediated hepatocellular apoptosis; 5) disorders of fatty acid metabolism; and 6) abnormal mitophagy. Thus, strategies to prevent mitochondrial damage or to manipulate mitochondrial function in a clinically useful manner may provide effective therapies to treat NAFLD. Natural products have been widely used to treat liver diseases, and have been reported as a highly important source for the development of promising drugs/nutrients for alleviating liver diseases. A rapidly expanding studies suggested that natural products affected mitochondrial function to remedy NAFLD [11–17], such as Shexiang Baoxin Pill, Punica granatum, Sida rhomboidea. Roxb, Cyclocarya paliurus, mangosteen pericarp, resveratrol and epigallocatechin gallate. Therefore, developing mitochondrial regulators/nutrients from natural products to remedy NAFLD-associated mitochondrial dysfunction represent attractive strategies for NAFLD therapy. Polygonati Rhizoma, first recorded in “Mingyi Bielu” (A.D. 220–450, Written by HongJing Tao), has been used as a traditional Chinese medicine and nutrient food for over 2000 years. Polygonatum kingianum Coll. et Hemsl., P. sibiricum Red. and P. cyrtonema Hua are described in Chinese Pharmacopoeia (2015 edition) as legal sources of Polygonati Rhizoma. P. kingianum (PK) are mainly distributed in the provinces of Yunnan, Sichuan, Guizhou and Guangxi in China. It has been reported that PK is mainly comprised of saponins and polysaccharides and has pharmacological activities, including immune promotion, anti-aging, blood glucose regulation, and lipid regulation [18,19]. In addition, our previous studies indicated that the total saponins and total polysaccharides from PK prevented type 2 diabetes [20,21]. However, it is not clear whether PK can alleviate NAFLD. Therefore, in this study, we investigated the alleviating effect and the underlying mechanism of action of PK on high-fat diet (HFD)-induced NAFLD. The results obtained indicated that PK alleviated HFD-induced NAFLD and multiple risk factors by promoting mitochondrial function. Taken together, PK may be a promising mitochondrial regulator/nutrient to remedy NAFLD-associated mitochondrial dysfunction and further alleviate NAFLD.
2.2. Preparation of PK extract The collected rhizome of PK was processed according to the regulation described in Chinese Pharmacopoeia (2015 edition). Briefly, fresh rhizome derived from PK was separated from fibrous roots, washed, cut into thick slices and dried in a dryer (YHG-S, Shanghai Yuejin Medical Instrument Factory, Shanghai, China) at 50 °C. Then, dried materials were infiltrated in a 5-fold volume of Shaoxing Rice Wine (Beijing Ershang Wangzhihe Food Co., Ltd., Beijing, China), and steamed in a steam sterilizer (LDZX-50 KBS, Shanghai Shenan Medical Instrument Factory, Shanghai, China) for 2.5 h at 120 °C. After cooling for 3 h, steamed materials were dried at 60 °C. Next, the steamed dried preparation of PK was pulverized, immersed in a seven-fold volume of water for 30 min and decocted with water for 60 min. Filtrates were collected after leaching. The dregs were successively decocted with a seven-fold volume of water for another 60 min, and the extracted liquids were filtered. The two successive filtrates were then combined and condensed using an RE-6000A rotatory evaporator (Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) under reduced pressure at 50 °C. Finally, the concentrates were lyophilized to a powder using a FD5-3 freeze dryer (SIM International Group Co. Ltd., Newark, DE, USA). Obtained powders of the extract were stored at room temperature in a desiccator until use.
2. Materials and methods 2.1. Chemicals, reagents, and materials Resveratrol was purchased from Chengdu Pufeide Biological Technology Co., Ltd. (Chengdu, China). Bicinchoninic Acid (BCA) Protein Determination Kit was provided by Beyotime Institute of Biotechnology (Shanghai, China). Basic basal rodent diet was obtained from Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd. (Suzhou, China). Cholesterol, refined lard, and eggs were provided by Beijing Boao Extension Co., Ltd. (Beijing, China), Sichuan Green Island Co., Ltd. (Chengdu, China) and Wal-Mart Supermarket (Kunming, China), respectively. High-purity deionized-water was purified by a Milli-Q System (Millipore, Bedford, MA, USA). Kits for the determination of alanine transaminase (ALT), aspartate transaminase
2.3. Animals and experimental design All procedures involving animals complied with the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health and were approved by the Institutional Ethical Committee on Animal Care and Experimentations of Yunnan University of Chinese Medicine (Kunming, China) (R-0620160026). All reasonable efforts were made to minimize the animals’ suffering. Healthy male Sprague–Dawley (SD) rats (180–200 g) were provided by DaShuo Biotech. Co., Ltd. (Chengdu, China). Rats were housed in an 2
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mitochondrial suspension was used for the determination of levels of MDA, SOD, GSH-PX, ATPase, and respiratory chain complex I and II using commercially available diagnostic kits in accordance with the manufacturer’s instructions on a SpectraMax Plus 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
environment at a temperature (24 ± 1 °C) and humidity (65 ± 10%) controlled animal room and maintained on a 12-h light/12-h dark cycle (light from 08:00 a.m. to 08:00 p.m.) with food and water provided ad libitum throughout the experiments. After one week of adaptive feeding, rats were randomized into the following seven groups (n = 7 rats per group): 1) normal control (NC) group (normal saline); 2) HFD group (normal saline); 3) resveratrol (RES) group (40 mg/kg); 4) low-dose PK (LPK) group (1 g/kg); 5) middle-dose PK (MPK) group (2 g/kg); and 6) high-dose PK (HPK) group (4 g/kg). Rats were intragastrically administered with the corresponding test compounds (or normal saline) once a day for 14 consecutive weeks. RES and PK were separately prepared in normal saline. NAFLD was induced by HFD (comprised of 1% cholesterol, 10% refined lard, 10% egg yolk, and 79% basic diet) for 14 consecutive weeks in all groups except for the normal control group.
2.9. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) Total RNA was extracted from liver tissue by trizol reagent, and was reverse transcribed into cDNA. A total of 2 μL cDNA template was used for PCR amplification using the following primers: UCP-2 target fragment upstream primer 5′-TCCCAATGTTGCCCGAAATG-3′, downstream primer 5′-TCGTCTGTCATGAGGTTGGC-3′, product length 99 bp; CPT-1 target fragment upstream primer 5′-ATGATCCCTCAGAGCCACAGC-3′, downstream primer 5′-TAGGTCTGCCGACACTTTGCC-3′, product length 91 bp; with stable expression of GAPDH as internal reference, target fragment upstream primer 5′-CTGGAGAAACCTGCCAAGT ATG-3′, downstream primer 5′- GGTGGAAGAATGGGAGTTGCT-3′, product length 138 bp. The total reaction volume was 25 μL. The PCR conditions were as follows: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s; annealing and extension for 15 s at 60 °C; and amplification for 40 cycles. The ratio of target products and internal reference GAPDH gray value (CT value) were calculated to reflect the relative mRNA expression levels of carnitine palmitoyl transferase-1 (CPT-1) and uncoupling protein-2 (UCP-2).
2.4. Sample collection During the experiment, the food intake of all rats was recorded daily, and body weight was recorded once every week. After the last administration in week 14, rats were fasted for 12 h and fasting blood was collected from the canthus. Then, rats were sacrificed and organs (liver, kidney and spleen) were harvested and stored at -80 ℃ until use. Blood samples were allowed to clot at 4 °C and centrifuged at 10,000×g for 10 min, after which serum was collected and stored at −80 °C until assayed. 2.5. Liver histological observation
2.10. Western blot analyses
Liver tissue from a portion of the left lobe was removed, fixed in 10% buffered formalin solution (pH 7.0) for at least 24 h, washed with PBS, dehydrated with gradient ethanol and transparentized with xylene, and embedded in paraffin. After hematoxylin and eosin (H&E) staining, histological parameters were observed under a light microscope and images were taken at 200×magnification.
Rat liver (100 mg) and mitochondria isolated from 100 mg of liver (which was used for detecting cytochrome c) were separately incubated for 30 min on ice with 1 mL RIPA lysis solution. Then, liver samples were homogenized using a glass homogenizer, and the lysate supernatants of liver and mitochondria were collected after centrifugation at 12,000g for 10 min at 4 °C. Protein concentrations were determined using the BCA method. A total of 40 μg protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on polyacrylamide gel (5% stacking gel at 80 V for 30 min and 12% separating gel at 120 V for 60 min) and transferred onto PVDF membranes using a transfer apparatus (Beijing Liuyi Biological Technology Co., Ltd., Beijing, China) at 300 mA for 65 min (30 min for cytochrome c). Nonspecific binding sites were blocked with 5% BSA in TBS-T (TBS plus 0.1% (v/v) Tween 20) for 2 h at room temperature with gentle rocking. Each membrane was then incubated with one of the following primary antibodies at 4 °C overnight: anti-Bcl-2 (at 1:1000 dilution), anti-Bax (at 1:8000 dilution), anti-Caspase-3 (at 1:2000 dilution), antiCaspase-9 (at 1:1000 dilution), anti-Cytchrome c (at 1:8000 dilution), and anti β-actin (at 1:4000 dilution). After three washes in TBS-T, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000 in 5% BSA/TBS-T) for 1 h at room temperature. Immunoreactive protein bands were visualized using a chemiluminescence HRP substrate using a ChemiDoc XRS image detector (Jena Analytical Instruments AG, Jena, Germany). β-actin was regarded as an internal reference. The intensity of protein bands was determined using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). The amount of protein under control conditions was assigned a relative value of 100%. Each band was normalized relative to the β-actin band in the same sample.
2.6. Determination of biochemical parameters of serum and liver A total of 100 μL of serum was used for measuring levels of ALT, AST, TG, TC, and LDL-C. Moreover, 100 μL of liver homogenate supernatant (in which the protein concentration was measured by BCA assay) was used for determining the levels of TG, TC, and HDL-C. All parameters were determined on a SpectraMax Plus 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using commercially available diagnostic kits (Nanjing Built Biological Engineering Research Institute, Nanjing, China) in accordance with the manufacturer’s instructions. 2.7. Isolation of liver mitochondria Liver mitochondria were isolated as previously described [22]. Briefly, rat liver (0.1 g) was placed into ice-cold isolation buffer (210 mM mannitol, 70 mM sucrose, 10 mM Tris base, 1 mM EDTA, and 0.5 mM EGTA, pH 7.4) to remove blood, and was minced into 1 mm3, then homogenized in isolation buffer using a Dounce glass homogenizer (Kimble/Kontes, Vineland, NJ, USA). After centrifugation at 1,000×g for 10 min, the supernatant was collected and centrifuged at 10,000×g for 10 min. The resulting precipitate was resuspended in isolation buffer and centrifuged at 10,000×g for 10 min to obtain a pellet containing liver mitochondria. All procedures were performed on ice or at 4 °C in a cold room.
2.11. Statistical processing 2.8. Evaluation of mitochondrial indicators involved in oxidative stress and energy metabolism
The data were expressed as the mean ± standard deviation (S.D.), and differences were considered significant when P < 0.05 or P < 0.01, as tested by one-way analysis of variance using SPSS version 21.0 (IBM, Armonk, NY, USA).
The liver mitochondria were resuspended in saline. After determining the protein concentration by BCA assay, 100 μL of 3
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Table 1 Body weight and food intake of rats.
Initial body weight (g) Final body weight (g) Body weight gain (g) Food intake (g)
NC
HFD
RES
LPK
MPK
HPK
199.14 ± 8.67 489.57 ± 36.09 290.43 ± 27.42 15934.24
200.86 ± 8.28 501.29 ± 38.32 300.43 ± 30.05 14863.94
204.57 ± 4.47 498.86 ± 26.62 294.29 ± 22.16 14359.24
201.57 ± 6.65 467.29 ± 9.20* 265.71 ± 2.54* 15324.26
201.14 ± 7.52 494.86 ± 15.10 293.71 ± 7.59 15431.22
198.71 ± 4.54 473.00 ± 22.78 274.29 ± 18.25 14596.47
Values represent the mean ± S.D. from 7 animals. HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; LPK, low-dose of Polygonatum kingianum; MPK, middle-dose of Polygonatum kingianum; NC; normal control; RES, resveratrol. * P < 0.05 versus the HFD group.
3. Results
3.3. Effects of HFD and PK on organ indexes
3.1. Effects of HFD and PK on body weight and food intake
After 14 weeks of HFD-feeding, the liver index, kidney index, and spleen index of rats in each group was calculated. As presented in Table 2, no significant differences were observed in the kidney and spleen index between rats in the NC and HFD group, indicating that the experimental method did not cause significant visceral index changes in rat kidney and spleen. However, when compared with the NC group, the liver index of rats in the HFD group increased significantly, indicating that 14 weeks of HFD feeding caused liver enlargement. However, after administering PK extract, the liver index of rats in the LPK and HPK group decreased significantly, demonstrating that PK effectively inhibited HFD-induced liver enlargement.
In our study, we used a HFD rat model to investigate the potential effect of PK on HFD-mediated NAFLD that was induced by administration of a HFD over a 14-week period. PK extract was administered by oral gavage at dosages of either 1, 2, or 4 g/kg/day during HFD administration. During the study, none of the rats died, they had healthylooking fur, normal drinking habits, moved freely and rapidly responded to external stimuli. As shown in Table 1, the HFD slightly increased body weight, whereas rats in the LPK treatment group showed significantly decreased body weight and weight gain without affecting food intake.
3.4. Effects of HFD and PK on liver lipid content 3.2. Effects of HFD and PK on serum parameters
Fig. 2A–C shows that after 14 weeks of HFD-feeding, TC and TG level were significantly increased with a notable decrease of HDL-C level, when compared with the NC group. However, these effects were significantly abolished by PK treatment. We also found that RES significantly decreased the levels of TC and TG. Furthermore, HE-staining revealed massive lipid accumulation (shown by arrowhead in Fig. 2D) in the HFD liver, and this accumulation was reduced by RES and PK treatment, resulting in a liver morphology that was similar to that of rats in the NC group (Fig. 2D).
In general, the HFD-induced NAFLD model is accompanied by hyperlipidemia and impaired hepatocytes. Fig. 1 shows that after 14 weeks of treatment, serum levels of TC, LDL-C, ALT, and AST were increased by HFD, and had no significant effect on the TG level. These effects were significantly restored by PK treatment. In addition, we found that RES significantly inhibited the increase in TC, LDL-C and AST level.
Fig. 1. Effects of HFD and PK on serum parameters. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFDfeeding, rats were sacrificed and, the serum was collected. Serum levels of (A) TC; (B) TG; (C) LDL-C; (D) ALT; and (E) AST were determined using commercial kits. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relative HFD group. ALT, alanine transaminase; AST, aspartate transaminase; HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; LDL-C, low density lipoprotein cholesterol; LPK, low-dose of Polygonatum kingianum; MPK, middle-dose of Polygonatum kingianum; NC; normal control; PK, Polygonatum kingianum; RES, resveratrol; SD, Sprague–Dawley; TC, total cholesterol; TG, triglyceride. 4
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Table 2 Effects of HFD and PK extract on organ indexes.
Body weight (g) Liver index (%) Kidney index (%) Spleen index (%)
NC
HFD
RES
LPK
MPK
HPK
489.57 ± 36.09 2.11 ± 0.19*** 0.48 ± 0.05 0.16 ± 0.02
501.29 ± 38.32 2.62 ± 0.33
498.86 ± 26.62 2.42 ± 0.12 0.51 ± 0.09 0.15 ± 0.01
467.29 ± 9.20 2.36 ± 0.15* 0.54 ± 0.04 0.15 ± 0.01
494.86 ± 15.10 2.53 ± 0.30 0.50 ± 0.02 0.15 ± 0.01
473.00 ± 22.78 2.36 ± 0.11* 0.51 ± 0.04 0.14 ± 0.01
0.16 ± 0.01
Values represent the mean ± S.D. from 7 animals. Organ indexes were calculated using the following formula: Organ index = [organ weight g / body weight g] × 100%. HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; LPK, low-dose of Polygonatum kingianum; MPK, middle-dose of Polygonatum kingianum; NC; normal control; RES, resveratrol. * P < 0.05. *** P < 0.001 versus the relative HFD group.
weeks of HFD-feeding, CPT-1 mRNA expression was significantly decreased, while UCP-2 mRNA expression was significantly up-regulated. However, these effects were significantly restored by treatment with PK extract. Furthermore, the decrease in CPT-1 mRNA expression level was remarkably restored by RES.
3.5. Effects of HFD and PK on mitochondrial status To investigate the involvement of mitochondria in HFD-induced oxidative stress, energy metabolism disorder, and NAFLD, several indicators were assessed in isolated liver mitochondria. As displayed in Fig. 3, induction of HFD significantly increased the MDA content and decreased activities of GSH-PX, SOD, Na+-K+-ATPase, and complex I and II in the liver mitochondria, with the no significant effect on Ca2+Mg2+-ATPase level. However, the effects were significantly restored after supplementation with PK extract. In addition, we found that RES significantly decreased the MDA level and increased activities of Na+K+-ATPase, and complex I and II.
3.7. Effects of HFD and PK on hepatocellular apoptosis To investigate the involvement of mitochondria in HFD-induced apoptosis and NAFLD, the expression of apoptosis-related proteins was evaluated. As shown in Fig. 5, induction of HFD significantly increased the expression of caspase 9, caspase 3, and Bax in hepatocytes, and decreased the expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. These effects were significantly restored by the treatment with PK extract and RES.
3.6. Effects of HFD and PK on fatty acid metabolism CPT-1 and UCP-2 are well-known regulators of fatty acid metabolism and mitochondrial biogenesis [23,24]. Fig. 4 shows that after 14
Fig. 2. Effects of HFD and PK on liver lipid content. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFDfeeding, rats were sacrificed, and the liver was collected. Liver levels of (A) TC; (B) TG; and (C) HDL-C were determined using commercial kits. Liver histology after HE-staining (D) was observed under a light microscope and images were taken at 100 × magnification. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group. HDL-C, high density lipoprotein cholesterol; HE, hematoxylin-eosin; →, lipid accumulation. 5
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Fig. 3. Effects of HFD and PK on mitochondrial status. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFDfeeding, rats were sacrificed, and the liver was collected for mitochondria isolation. Levels of (A) MDA; (B) GSH-PX; (C) SOD; (D) Na+-K+-ATPase; (E) Ca2+-Mg2+ATPase; (F) Complex I; and (G) Complex II in isolated liver mitochondria were determined using commercial kits. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group. ATPase, ATP synthase; GSH-PX, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase.
4. Discussion
level and liver enlargement, indicating that HFD induces hyperlipidemia and impaired hepatocytes. However, PK extract significantly inhibited these HFD-induced changes without affecting food intake, indicating that PK effectively regulated liver lipid metabolism disorders, and thereby had an alleviating effect on HFD-induced NAFLD rats. Mitochondria are important organelles inside eukaryotic cells, and serve as the cellular energy supply center. The Krebs cycle, β-oxidation of fatty acid, oxidative phosphorylation, and the urea cycle are all conducted inside mitochondria [29]. Mitochondrial dysfunction leads to the excessive production of reactive oxygen species (ROS), and is the major source of ROS [30]. ROS can attack polyunsaturated fatty acids in membrane phospholipids, thereby producing large amounts of lipid peroxides (MDA), which is used as an index that reflects the oxidative status [31]. Additionally, SOD and GSH-PX, vital scavengers of ROS in the body [32], can inhibit lipid peroxidation. When hepatocytes are
NAFLD, which is closely associated with metabolic pathologies, is the most common chronic liver disease worldwide. Alarming epidemics of metabolic syndrome and obesity have accelerated the global prevalence of NAFLD [25]. Therefore, in the present study, an HFD-induced NAFLD model was established to study the alleviating effects of PK extract and its underlying mechanism of action, with RES as the positive control (because RES can effectively regulate lipid metabolism and mitochondrial function to alleviate NAFLD [26,27]). NAFLD is associated with an excessive lipid accumulation in the liver (which is beyond the scavenging ability of liver), and is mainly characterized by increases in TC, TG, and LDL-C content in the serum and/or liver, and a reduced HDL-C content [28]. We found that HFD-feeding resulted in notable increases in ALT, AST, TC, and LDL-C level in serum and TC and TG level in the liver. In addition, HFD-feeding results in reduced HDL-C
Fig. 4. Effects of HFD and PK on fatty acid metabolism. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFD-feeding, rats were sacrificed, and the liver was collected. Expression levels of (A) CPT-1 mRNA and (B) UCP-2 mRNA were evaluated using real-time quantitative reverse transcription polymerase chain reaction. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group. CPT-1, Carnitine palmitoyltransferase-1; UCP2, uncoupling protein-2.
6
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Fig. 5. Effects of HFD and PK on hepatocellular apoptosis. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/ kg/day). After 14 weeks of HFD-feeding, rats were sacrificed, and the liver was collected. Hepatocellular mitochondria were isolated for determining the expression of cytchrome c. Expression levels of (B) caspase-9 (B1, 37 KD; B2, 50 KD), (C) caspase-3 (C1, 32 KD; C2, 17 KD), (D) Bcl-2, (E) Bax, (F) cytochrome c were evaluated by Western blot analyses. Immunoreactive protein bands (A) were visualized with a chemiluminescence horseradish peroxidase substrate. Values represent the mean ± S.D. from 6 randomly-selected animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group.
oxidation of short-chain (< C8), medium-chain (C8–C12), and longchain (C12–C20) fatty acids [34]. Short- and medium-chain fatty acids can freely enter the mitochondria, while long-chain fatty acids only enter the mitochondria with the assistance of CPT-1 transport [23]. Thus, mitochondrial CPT-1 is the primary rate-limiting enzyme of βoxidation. Increasing the activity or expression of CPT-1 will facilitate the oxidation of fatty acids accumulated in the liver, which is beneficial for alleviating NAFLD. We demonstrated that HFD-feeding significantly down-regulated the expression of CPT-1 mRNA in the liver, which was significantly inverted by treatment with PK extract, demonstrating that PK accelerated beta-oxidation of fatty acid within the liver to protect HFD-induced NAFLD rats. In previous studies, it was demonstrated that apoptosis aggravated hepatocellular injury. The release of cytochrome c is a critical step in the apoptotic process [35,36]. Cytochrome c is released into the cytoplasm and binds to apoptotic proteases, thereby activating factor-1 (Apaf-1) to form a polymer in the presence of deoxyadenosine triphosphate (dATP), which promotes its binding with caspase precursor protein (pro-caspase) to form apoptotic bodies and further activate caspase-9. Activated caspase-9 can activate other caspases (e.x. caspase3), which results in apoptosis. Bax, one of the earliest pro-apoptotic proteins discovered, mainly resides in the cytoplasm in normal cells. However, upon stimulation by apoptotic signals, it can be translocated to mitochondria, which causes the release of cytochrome c into the cytoplasm [37–39]. Bcl-2 is the main anti-apoptotic protein in its family of proteins and contains four domains (BH1 to BH4) that stabilize mitochondrial membrane function and prevents mitochondria releasing apoptotic factors, such as apoptosis-inducing factor and cytochrome c [39,40]. We found that HFD-feeding significantly increased the expression of caspase 9, caspase 3 and Bax in hepatocytes, and decreased the expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. These effects were significantly inverted by PK treatment, indicating that PK inhibited hepatocellular apoptosis and decreased liver injury to alleviate HFD-induced NAFLD. In addition, it is clearly noted that the effects of three doses of PK
excessively attacked by ROS, their levels may be decreased. Therefore, determining MDA, SOD, and GSH-PX levels not only reflect the degree of liver damage, but also reflect the effect of drugs on the oxidative stress status in hepatocytes. We found that HFD-feeding led to a significant increase in MDA and reduced SOD and GSH-PX level in liver mitochondria from NAFLD rats. However, these effects were significantly restored by PK treatment, suggesting that PK may enhance the body's ability to scavenge ROS from mitochondria and further alleviate its lipid peroxidation of cell membranes to exert a protective effect on NAFLD rats. ATP, as a major energy substance in the body, is mainly synthesized in mitochondria, and depends on the activity of complexes on the mitochondrial respiratory chain [29]. When the function of the respiratory chain is damaged, energy metabolism will be prevented. This effect will decrease the ability of hepatocytes to treat and transport liver lipids, which may result in lipid accumulation within the liver and further aggravate HFD-induced NAFLD. In addition, lipid accumulation in the liver will up-regulate UCP-2 expression and further decrease ATP level, which not only decreases energy supply of TC synthesis and further inhibits lipid formation, but also promotes the β-oxidation of fatty acids and further alleviates liver lipid accumulation [24]. However, excessive UCP-2 expression will decrease the ATP reserve in mitochondria [24,33], which aggravates liver injury when the ATP requirement of hepatocytes is significantly increased. In this study, it was found that the activities of ATPase, and complex I and II in the liver mitochondria were reduced by HFD-feeding, with the increasing of UCP-2 mRNA expression in liver, whereas the effects were inverted by treatment with PK supplement, indicating that PK may promote oxidation rate of liver respiratory chain and decrease oxidative phosphorylation uncoupling, which further enhances mitochondrial energy metabolism to alleviate HFD-induced NAFLD. Lipid metabolism disorder can increase the supply of fatty acids in the liver, resulting in the accumulation of fatty acids in the liver, leading to NAFLD. Mitochondrial β-oxidation is the main oxidative pathway for the disposal of fatty acids and primarily involves the 7
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Fig. 6. The regulation of PK extract on HFD-induced NAFLD.
Acknowledgments
extract on the assayed indicators did not show dose-dependent manners, which was similar to many other herbal medicines [41,42]. This may be attributed to the complex constituents and pharmacological mechanism of the herbs (e.g., a mode of action of multi-compound, multi-pathway and multi-target [43]). Besides, it is very likely that the PK dosages used in our study were not in the range of displaying dosedependence.
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5. Conclusion Taken together, our data indicated that PK alleviated HFD-induced NAFLD and multiple risk factors by promoting mitochondrial functions, which may be related to the enhancement of mitochondrial anti-oxidative status, energy metabolism and beta-oxidation of fatty acid, as well as inhibition of hepatocellular apoptosis (Fig. 6). PK may be used for developing mitochondrial regulators/nutrients to remedy NAFLDassociated mitochondrial dysfunction to further alleviate NAFLD. Further investigation regarding the effects of PK on the absorption and excretion of lipids remains of investigative interest. Author contributions Xing-Xin Yang and Xi Wang wrote the manuscript. Xi Wang, XingXin Yang, Jin-Cai Dong and Feng-Jiao Li conducted the experiments. Ting-Ting Shi provided technical support and helpful discussions. Lin-Xi Zeng, Min Yang, Wen Gu and Jing-Ping Li participated in writing and modifying the manuscript. Jie Yu and Xing-Xin Yang designed the study. All authors have no conflicts of interest. Ethics approval and consent to participate Approval from the Institutional Ethical Committee on Animal Care and Experimentations of Yunnan University of Chinese Medicine (R0620160026) was obtained for this study. Funding This study was supported by grants from the National Natural Science Foundation of China (Grants 81660596, 81460623 and 81760733), and the Application and Basis Research Project of Yunnan China (Grants 2016FD050 and 2017FF117-013). Availability of data and materials All data used to support the findings of this study are available from the corresponding author upon request. Consent for publication Publication of the manuscript has been approved by all co-authors. 8
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Mitochondrial dysfunction in high-fat diet-induced nonalcoholic fatty liver disease: The alleviating effect and its mechanism of Polygonatum kingianum
T
Xing-Xin Yanga,b,1, Xi Wanga,b,1, Ting-Ting Shic, Jin-Cai Donga,b, Feng-Jiao Lia,b, Lin-Xi Zenga,b, ⁎ Min Yanga,b, Wen Gua,b, Jing-Ping Lia,b, Jie Yua,b, a
College of Pharmaceutical Science, Yunnan University of Chinese Medicine, 1076 Yuhua Road, Kunming, 650500, China Kunming Key Laboratory for Metabolic Diseases Prevention and Treatment by Chinese Medicine, 1076 Yuhua Road, Kunming, 650500, China c Department of Pharmaceutical Preparation, The Xixi Hospital of Hangzhou Affiliated to Zhejiang University of Traditional Chinese medicine, Hangzhou, 310023, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: High-fat diet Mechanism Mitochondria Nonalcoholic Fatty liver Polygonatum kingianum Protective effect
Background: Mitochondrial dysfunction is an important mechanism of non-alcoholic fatty liver disease (NAFLD). Developing mitochondrial regulators/nutrients from natural products to remedy mitochondrial dysfunction represent attractive strategies for NAFLD therapy. In China, Polygonatum kingianum (PK) has been used as a herb and food nutrient for centuries. So far, studies in which the effects of PK on NAFLD are evaluated are lacking. Our study aims at identifying the effects and mechanism of action of PK on NAFLD based on mitochondrial regulation. Methods: A NAFLD rat model was induced by a high-fat diet (HFD) and rats were intragastrically given PK (1, 2 and 4 g/kg) for 14 weeks. Changes in body weight, food intake, histological parameters, organ indexes, biochemical parameters and mitochondrial indicators involved in oxidative stress, energy metabolism, fatty acid metabolism, and apoptosis were investigated. Results: PK significantly inhibited the HFD-induced increase of alanine transaminase, aspartate transaminase, total cholesterol (TC), and low density lipoprotein cholesterol in serum, and TC and triglyceride in the liver. In addition, PK reduced high density lipoprotein cholesterol and liver enlargement without affecting food intake. PK also remarkably inhibited the HFD-induced increase of malondialdehyde and the reduction of superoxide dismutase, glutathione peroxidase, ATP synthase, and complex I and II, in mitochondria. Moreover, mRNA expression of carnitine palmitoyl transferase-1 and uncoupling protein-2 was significantly up-regulated and down-regulated after PK treatment, respectively. Finally, PK notably inhibited the HFD-induced increase of caspase 9, caspase 3 and Bax expression in hepatocytes, and the decrease of expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. Conclusion: PK alleviated HFD-induced NAFLD by promoting mitochondrial functions. Thus, PK may be useful mitochondrial regulators/nutrients to remedy mitochondrial dysfunction and alleviate NAFLD.
1. Introduction Non-alcoholic fatty liver disease (NAFLD), which is characterized as an abnormal accumulation of triglycerides in hepatocytes, isnow considered the most prevalent type of chronic liver disease worldwide with
an incidence that is even higher with respect to liver damage caused by hepatitis C virus or alcohol abuse [1]. NAFLD is a histological disease, which may progress from simple steatosis to nonalcoholic steatohepatitis, liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma [2,3].
Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; ATPase, ATP synthase; BCA, bicinchoninic acid; CPT-1, carnitine palmitoyl transferase-1; GSH-PX, glutathione peroxidase; HDL-C, high density lipoprotein cholesterol; HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; HRP, horseradishperoxidase; LDL-C, low density lipoprotein cholesterol; LPK, low-dose of Polygonatum kingianum; MDA, malondialdehyde; MPK, middle-dose of Polygonatum kingianum; NAFLD, non-alcoholic fatty liver disease; NC, normal control; PK, polygonatum kingianum; RES, resveratrol; ROS, reactive oxygen species; RT-PCR, real-time quantitative reverse transcription polymerase chain reaction; SD, sprague–Dawley; SOD, superoxide dismutase; TC, total cholesterol; TG, triglyceride; UCP-2, uncoupling protein-2 ⁎ Corresponding author at: College of Pharmaceutical Science, Yunnan University of Chinese Medicine, 1076 Yuhua Road, Kunming, 650500, China. E-mail address: [email protected] (J. Yu). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109083 Received 16 February 2019; Received in revised form 3 June 2019; Accepted 3 June 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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(AST), triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and ATP synthase (ATPase) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). In addition, kits to determine respiratory chain complex I and II were provided by Genmed Scientifics Inc. (Arlington, MA, USA). Bcl-2 mouse monoclonal antibody was purchased from Shanghai Jiwei Biological Technology Co., Ltd. (Shanghai, China). Cytchrome c mouse monoclonal antibody, β-actin mouse monoclonal antibody, horseradish-peroxidase (HRP) AffiniPure Goat Anti-Mouse IgG (H + L) antibody and HRP AffiniPure Goat Anti-Rabbit IgG (H + L) antibody were purchased from the Proteintech Group (Chicago, IL, USA). Caspase 3 rabbit monoclonal antibody, caspase 9 rabbit monoclonal antibody, and Bax rabbit monoclonal antibody were purchased from Abcam (Cambridge, UK). Primers were designed by Servicebio (Wuhan, China). TRIzol®Reagent was purchased from Ambion (Carlsbad, CA, USA). RevertAid First Strand cDNA Synthesis Kit was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA), and FastStart Universal SYBR Green Master (Rox) was purchased from Roche (Basel, Switzerland). Highly Efficient RIPA Tissue/Cell Lysate was provided by Solarbio Science & Technology Co., Ltd. (Beijing, China). Immobilon™ Western Chemiluminescence HRP Substrate was obtained from Millipore Corporation (Bedford, MA, USA). All other reagents were of analytical grade or higher. The rhizome of PK (purchase date April 07, 2017) was purchased from Wenshan Shengnong Trueborn Medicinal Materials Cultivation Cooperation Society (Wenshan, China). Samples were authenticated by Professor Jie Yu, and a voucher specimen of PK (No. 8426) was deposited in the Key Laboratory of Preventing Metabolic Diseases of Traditional Chinese Medicine, Yunnan University of Chinese Medicine (Kunming, China).
To date, no approved drugs are available to treat patients with fatty liver disease and clinical management strategies mainly rely on modifications of diet, physical activity, and lifestyle, as well as on correcting hyperglycemia, insulin resistance, and hyperlipidemia, which are metabolic disturbances associated with NAFLD [3]. Therefore, it is of great value to develop treatment regimens and identify drugs/nutrients to alleviate NAFLD. Mitochondria are play a central role in complex processes, including the generation of energy and reactive oxygen species (ROS), maintaining calcium homeostasis, and adjusting apoptosis and lipid metabolism [4]. In an increasing number of studies, it has been demonstrated that mitochondrial dysfunction is an important mechanism of NAFLD and its aggravation and in which the following mechanisms may play a role [5–12]: 1) mitochondrial DNA damage; 2) disorders of energy metabolism; 3) oxidative stress and lipid peroxidation; 4) mitochondria-mediated hepatocellular apoptosis; 5) disorders of fatty acid metabolism; and 6) abnormal mitophagy. Thus, strategies to prevent mitochondrial damage or to manipulate mitochondrial function in a clinically useful manner may provide effective therapies to treat NAFLD. Natural products have been widely used to treat liver diseases, and have been reported as a highly important source for the development of promising drugs/nutrients for alleviating liver diseases. A rapidly expanding studies suggested that natural products affected mitochondrial function to remedy NAFLD [11–17], such as Shexiang Baoxin Pill, Punica granatum, Sida rhomboidea. Roxb, Cyclocarya paliurus, mangosteen pericarp, resveratrol and epigallocatechin gallate. Therefore, developing mitochondrial regulators/nutrients from natural products to remedy NAFLD-associated mitochondrial dysfunction represent attractive strategies for NAFLD therapy. Polygonati Rhizoma, first recorded in “Mingyi Bielu” (A.D. 220–450, Written by HongJing Tao), has been used as a traditional Chinese medicine and nutrient food for over 2000 years. Polygonatum kingianum Coll. et Hemsl., P. sibiricum Red. and P. cyrtonema Hua are described in Chinese Pharmacopoeia (2015 edition) as legal sources of Polygonati Rhizoma. P. kingianum (PK) are mainly distributed in the provinces of Yunnan, Sichuan, Guizhou and Guangxi in China. It has been reported that PK is mainly comprised of saponins and polysaccharides and has pharmacological activities, including immune promotion, anti-aging, blood glucose regulation, and lipid regulation [18,19]. In addition, our previous studies indicated that the total saponins and total polysaccharides from PK prevented type 2 diabetes [20,21]. However, it is not clear whether PK can alleviate NAFLD. Therefore, in this study, we investigated the alleviating effect and the underlying mechanism of action of PK on high-fat diet (HFD)-induced NAFLD. The results obtained indicated that PK alleviated HFD-induced NAFLD and multiple risk factors by promoting mitochondrial function. Taken together, PK may be a promising mitochondrial regulator/nutrient to remedy NAFLD-associated mitochondrial dysfunction and further alleviate NAFLD.
2.2. Preparation of PK extract The collected rhizome of PK was processed according to the regulation described in Chinese Pharmacopoeia (2015 edition). Briefly, fresh rhizome derived from PK was separated from fibrous roots, washed, cut into thick slices and dried in a dryer (YHG-S, Shanghai Yuejin Medical Instrument Factory, Shanghai, China) at 50 °C. Then, dried materials were infiltrated in a 5-fold volume of Shaoxing Rice Wine (Beijing Ershang Wangzhihe Food Co., Ltd., Beijing, China), and steamed in a steam sterilizer (LDZX-50 KBS, Shanghai Shenan Medical Instrument Factory, Shanghai, China) for 2.5 h at 120 °C. After cooling for 3 h, steamed materials were dried at 60 °C. Next, the steamed dried preparation of PK was pulverized, immersed in a seven-fold volume of water for 30 min and decocted with water for 60 min. Filtrates were collected after leaching. The dregs were successively decocted with a seven-fold volume of water for another 60 min, and the extracted liquids were filtered. The two successive filtrates were then combined and condensed using an RE-6000A rotatory evaporator (Shanghai Yarong Biochemical Instrument Factory, Shanghai, China) under reduced pressure at 50 °C. Finally, the concentrates were lyophilized to a powder using a FD5-3 freeze dryer (SIM International Group Co. Ltd., Newark, DE, USA). Obtained powders of the extract were stored at room temperature in a desiccator until use.
2. Materials and methods 2.1. Chemicals, reagents, and materials Resveratrol was purchased from Chengdu Pufeide Biological Technology Co., Ltd. (Chengdu, China). Bicinchoninic Acid (BCA) Protein Determination Kit was provided by Beyotime Institute of Biotechnology (Shanghai, China). Basic basal rodent diet was obtained from Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd. (Suzhou, China). Cholesterol, refined lard, and eggs were provided by Beijing Boao Extension Co., Ltd. (Beijing, China), Sichuan Green Island Co., Ltd. (Chengdu, China) and Wal-Mart Supermarket (Kunming, China), respectively. High-purity deionized-water was purified by a Milli-Q System (Millipore, Bedford, MA, USA). Kits for the determination of alanine transaminase (ALT), aspartate transaminase
2.3. Animals and experimental design All procedures involving animals complied with the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health and were approved by the Institutional Ethical Committee on Animal Care and Experimentations of Yunnan University of Chinese Medicine (Kunming, China) (R-0620160026). All reasonable efforts were made to minimize the animals’ suffering. Healthy male Sprague–Dawley (SD) rats (180–200 g) were provided by DaShuo Biotech. Co., Ltd. (Chengdu, China). Rats were housed in an 2
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mitochondrial suspension was used for the determination of levels of MDA, SOD, GSH-PX, ATPase, and respiratory chain complex I and II using commercially available diagnostic kits in accordance with the manufacturer’s instructions on a SpectraMax Plus 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).
environment at a temperature (24 ± 1 °C) and humidity (65 ± 10%) controlled animal room and maintained on a 12-h light/12-h dark cycle (light from 08:00 a.m. to 08:00 p.m.) with food and water provided ad libitum throughout the experiments. After one week of adaptive feeding, rats were randomized into the following seven groups (n = 7 rats per group): 1) normal control (NC) group (normal saline); 2) HFD group (normal saline); 3) resveratrol (RES) group (40 mg/kg); 4) low-dose PK (LPK) group (1 g/kg); 5) middle-dose PK (MPK) group (2 g/kg); and 6) high-dose PK (HPK) group (4 g/kg). Rats were intragastrically administered with the corresponding test compounds (or normal saline) once a day for 14 consecutive weeks. RES and PK were separately prepared in normal saline. NAFLD was induced by HFD (comprised of 1% cholesterol, 10% refined lard, 10% egg yolk, and 79% basic diet) for 14 consecutive weeks in all groups except for the normal control group.
2.9. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) Total RNA was extracted from liver tissue by trizol reagent, and was reverse transcribed into cDNA. A total of 2 μL cDNA template was used for PCR amplification using the following primers: UCP-2 target fragment upstream primer 5′-TCCCAATGTTGCCCGAAATG-3′, downstream primer 5′-TCGTCTGTCATGAGGTTGGC-3′, product length 99 bp; CPT-1 target fragment upstream primer 5′-ATGATCCCTCAGAGCCACAGC-3′, downstream primer 5′-TAGGTCTGCCGACACTTTGCC-3′, product length 91 bp; with stable expression of GAPDH as internal reference, target fragment upstream primer 5′-CTGGAGAAACCTGCCAAGT ATG-3′, downstream primer 5′- GGTGGAAGAATGGGAGTTGCT-3′, product length 138 bp. The total reaction volume was 25 μL. The PCR conditions were as follows: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s; annealing and extension for 15 s at 60 °C; and amplification for 40 cycles. The ratio of target products and internal reference GAPDH gray value (CT value) were calculated to reflect the relative mRNA expression levels of carnitine palmitoyl transferase-1 (CPT-1) and uncoupling protein-2 (UCP-2).
2.4. Sample collection During the experiment, the food intake of all rats was recorded daily, and body weight was recorded once every week. After the last administration in week 14, rats were fasted for 12 h and fasting blood was collected from the canthus. Then, rats were sacrificed and organs (liver, kidney and spleen) were harvested and stored at -80 ℃ until use. Blood samples were allowed to clot at 4 °C and centrifuged at 10,000×g for 10 min, after which serum was collected and stored at −80 °C until assayed. 2.5. Liver histological observation
2.10. Western blot analyses
Liver tissue from a portion of the left lobe was removed, fixed in 10% buffered formalin solution (pH 7.0) for at least 24 h, washed with PBS, dehydrated with gradient ethanol and transparentized with xylene, and embedded in paraffin. After hematoxylin and eosin (H&E) staining, histological parameters were observed under a light microscope and images were taken at 200×magnification.
Rat liver (100 mg) and mitochondria isolated from 100 mg of liver (which was used for detecting cytochrome c) were separately incubated for 30 min on ice with 1 mL RIPA lysis solution. Then, liver samples were homogenized using a glass homogenizer, and the lysate supernatants of liver and mitochondria were collected after centrifugation at 12,000g for 10 min at 4 °C. Protein concentrations were determined using the BCA method. A total of 40 μg protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on polyacrylamide gel (5% stacking gel at 80 V for 30 min and 12% separating gel at 120 V for 60 min) and transferred onto PVDF membranes using a transfer apparatus (Beijing Liuyi Biological Technology Co., Ltd., Beijing, China) at 300 mA for 65 min (30 min for cytochrome c). Nonspecific binding sites were blocked with 5% BSA in TBS-T (TBS plus 0.1% (v/v) Tween 20) for 2 h at room temperature with gentle rocking. Each membrane was then incubated with one of the following primary antibodies at 4 °C overnight: anti-Bcl-2 (at 1:1000 dilution), anti-Bax (at 1:8000 dilution), anti-Caspase-3 (at 1:2000 dilution), antiCaspase-9 (at 1:1000 dilution), anti-Cytchrome c (at 1:8000 dilution), and anti β-actin (at 1:4000 dilution). After three washes in TBS-T, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000 in 5% BSA/TBS-T) for 1 h at room temperature. Immunoreactive protein bands were visualized using a chemiluminescence HRP substrate using a ChemiDoc XRS image detector (Jena Analytical Instruments AG, Jena, Germany). β-actin was regarded as an internal reference. The intensity of protein bands was determined using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). The amount of protein under control conditions was assigned a relative value of 100%. Each band was normalized relative to the β-actin band in the same sample.
2.6. Determination of biochemical parameters of serum and liver A total of 100 μL of serum was used for measuring levels of ALT, AST, TG, TC, and LDL-C. Moreover, 100 μL of liver homogenate supernatant (in which the protein concentration was measured by BCA assay) was used for determining the levels of TG, TC, and HDL-C. All parameters were determined on a SpectraMax Plus 384 Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) using commercially available diagnostic kits (Nanjing Built Biological Engineering Research Institute, Nanjing, China) in accordance with the manufacturer’s instructions. 2.7. Isolation of liver mitochondria Liver mitochondria were isolated as previously described [22]. Briefly, rat liver (0.1 g) was placed into ice-cold isolation buffer (210 mM mannitol, 70 mM sucrose, 10 mM Tris base, 1 mM EDTA, and 0.5 mM EGTA, pH 7.4) to remove blood, and was minced into 1 mm3, then homogenized in isolation buffer using a Dounce glass homogenizer (Kimble/Kontes, Vineland, NJ, USA). After centrifugation at 1,000×g for 10 min, the supernatant was collected and centrifuged at 10,000×g for 10 min. The resulting precipitate was resuspended in isolation buffer and centrifuged at 10,000×g for 10 min to obtain a pellet containing liver mitochondria. All procedures were performed on ice or at 4 °C in a cold room.
2.11. Statistical processing 2.8. Evaluation of mitochondrial indicators involved in oxidative stress and energy metabolism
The data were expressed as the mean ± standard deviation (S.D.), and differences were considered significant when P < 0.05 or P < 0.01, as tested by one-way analysis of variance using SPSS version 21.0 (IBM, Armonk, NY, USA).
The liver mitochondria were resuspended in saline. After determining the protein concentration by BCA assay, 100 μL of 3
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Table 1 Body weight and food intake of rats.
Initial body weight (g) Final body weight (g) Body weight gain (g) Food intake (g)
NC
HFD
RES
LPK
MPK
HPK
199.14 ± 8.67 489.57 ± 36.09 290.43 ± 27.42 15934.24
200.86 ± 8.28 501.29 ± 38.32 300.43 ± 30.05 14863.94
204.57 ± 4.47 498.86 ± 26.62 294.29 ± 22.16 14359.24
201.57 ± 6.65 467.29 ± 9.20* 265.71 ± 2.54* 15324.26
201.14 ± 7.52 494.86 ± 15.10 293.71 ± 7.59 15431.22
198.71 ± 4.54 473.00 ± 22.78 274.29 ± 18.25 14596.47
Values represent the mean ± S.D. from 7 animals. HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; LPK, low-dose of Polygonatum kingianum; MPK, middle-dose of Polygonatum kingianum; NC; normal control; RES, resveratrol. * P < 0.05 versus the HFD group.
3. Results
3.3. Effects of HFD and PK on organ indexes
3.1. Effects of HFD and PK on body weight and food intake
After 14 weeks of HFD-feeding, the liver index, kidney index, and spleen index of rats in each group was calculated. As presented in Table 2, no significant differences were observed in the kidney and spleen index between rats in the NC and HFD group, indicating that the experimental method did not cause significant visceral index changes in rat kidney and spleen. However, when compared with the NC group, the liver index of rats in the HFD group increased significantly, indicating that 14 weeks of HFD feeding caused liver enlargement. However, after administering PK extract, the liver index of rats in the LPK and HPK group decreased significantly, demonstrating that PK effectively inhibited HFD-induced liver enlargement.
In our study, we used a HFD rat model to investigate the potential effect of PK on HFD-mediated NAFLD that was induced by administration of a HFD over a 14-week period. PK extract was administered by oral gavage at dosages of either 1, 2, or 4 g/kg/day during HFD administration. During the study, none of the rats died, they had healthylooking fur, normal drinking habits, moved freely and rapidly responded to external stimuli. As shown in Table 1, the HFD slightly increased body weight, whereas rats in the LPK treatment group showed significantly decreased body weight and weight gain without affecting food intake.
3.4. Effects of HFD and PK on liver lipid content 3.2. Effects of HFD and PK on serum parameters
Fig. 2A–C shows that after 14 weeks of HFD-feeding, TC and TG level were significantly increased with a notable decrease of HDL-C level, when compared with the NC group. However, these effects were significantly abolished by PK treatment. We also found that RES significantly decreased the levels of TC and TG. Furthermore, HE-staining revealed massive lipid accumulation (shown by arrowhead in Fig. 2D) in the HFD liver, and this accumulation was reduced by RES and PK treatment, resulting in a liver morphology that was similar to that of rats in the NC group (Fig. 2D).
In general, the HFD-induced NAFLD model is accompanied by hyperlipidemia and impaired hepatocytes. Fig. 1 shows that after 14 weeks of treatment, serum levels of TC, LDL-C, ALT, and AST were increased by HFD, and had no significant effect on the TG level. These effects were significantly restored by PK treatment. In addition, we found that RES significantly inhibited the increase in TC, LDL-C and AST level.
Fig. 1. Effects of HFD and PK on serum parameters. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFDfeeding, rats were sacrificed and, the serum was collected. Serum levels of (A) TC; (B) TG; (C) LDL-C; (D) ALT; and (E) AST were determined using commercial kits. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relative HFD group. ALT, alanine transaminase; AST, aspartate transaminase; HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; LDL-C, low density lipoprotein cholesterol; LPK, low-dose of Polygonatum kingianum; MPK, middle-dose of Polygonatum kingianum; NC; normal control; PK, Polygonatum kingianum; RES, resveratrol; SD, Sprague–Dawley; TC, total cholesterol; TG, triglyceride. 4
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Table 2 Effects of HFD and PK extract on organ indexes.
Body weight (g) Liver index (%) Kidney index (%) Spleen index (%)
NC
HFD
RES
LPK
MPK
HPK
489.57 ± 36.09 2.11 ± 0.19*** 0.48 ± 0.05 0.16 ± 0.02
501.29 ± 38.32 2.62 ± 0.33
498.86 ± 26.62 2.42 ± 0.12 0.51 ± 0.09 0.15 ± 0.01
467.29 ± 9.20 2.36 ± 0.15* 0.54 ± 0.04 0.15 ± 0.01
494.86 ± 15.10 2.53 ± 0.30 0.50 ± 0.02 0.15 ± 0.01
473.00 ± 22.78 2.36 ± 0.11* 0.51 ± 0.04 0.14 ± 0.01
0.16 ± 0.01
Values represent the mean ± S.D. from 7 animals. Organ indexes were calculated using the following formula: Organ index = [organ weight g / body weight g] × 100%. HFD, high-fat diet; HPK, high-dose of Polygonatum kingianum; LPK, low-dose of Polygonatum kingianum; MPK, middle-dose of Polygonatum kingianum; NC; normal control; RES, resveratrol. * P < 0.05. *** P < 0.001 versus the relative HFD group.
weeks of HFD-feeding, CPT-1 mRNA expression was significantly decreased, while UCP-2 mRNA expression was significantly up-regulated. However, these effects were significantly restored by treatment with PK extract. Furthermore, the decrease in CPT-1 mRNA expression level was remarkably restored by RES.
3.5. Effects of HFD and PK on mitochondrial status To investigate the involvement of mitochondria in HFD-induced oxidative stress, energy metabolism disorder, and NAFLD, several indicators were assessed in isolated liver mitochondria. As displayed in Fig. 3, induction of HFD significantly increased the MDA content and decreased activities of GSH-PX, SOD, Na+-K+-ATPase, and complex I and II in the liver mitochondria, with the no significant effect on Ca2+Mg2+-ATPase level. However, the effects were significantly restored after supplementation with PK extract. In addition, we found that RES significantly decreased the MDA level and increased activities of Na+K+-ATPase, and complex I and II.
3.7. Effects of HFD and PK on hepatocellular apoptosis To investigate the involvement of mitochondria in HFD-induced apoptosis and NAFLD, the expression of apoptosis-related proteins was evaluated. As shown in Fig. 5, induction of HFD significantly increased the expression of caspase 9, caspase 3, and Bax in hepatocytes, and decreased the expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. These effects were significantly restored by the treatment with PK extract and RES.
3.6. Effects of HFD and PK on fatty acid metabolism CPT-1 and UCP-2 are well-known regulators of fatty acid metabolism and mitochondrial biogenesis [23,24]. Fig. 4 shows that after 14
Fig. 2. Effects of HFD and PK on liver lipid content. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFDfeeding, rats were sacrificed, and the liver was collected. Liver levels of (A) TC; (B) TG; and (C) HDL-C were determined using commercial kits. Liver histology after HE-staining (D) was observed under a light microscope and images were taken at 100 × magnification. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group. HDL-C, high density lipoprotein cholesterol; HE, hematoxylin-eosin; →, lipid accumulation. 5
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Fig. 3. Effects of HFD and PK on mitochondrial status. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFDfeeding, rats were sacrificed, and the liver was collected for mitochondria isolation. Levels of (A) MDA; (B) GSH-PX; (C) SOD; (D) Na+-K+-ATPase; (E) Ca2+-Mg2+ATPase; (F) Complex I; and (G) Complex II in isolated liver mitochondria were determined using commercial kits. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group. ATPase, ATP synthase; GSH-PX, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase.
4. Discussion
level and liver enlargement, indicating that HFD induces hyperlipidemia and impaired hepatocytes. However, PK extract significantly inhibited these HFD-induced changes without affecting food intake, indicating that PK effectively regulated liver lipid metabolism disorders, and thereby had an alleviating effect on HFD-induced NAFLD rats. Mitochondria are important organelles inside eukaryotic cells, and serve as the cellular energy supply center. The Krebs cycle, β-oxidation of fatty acid, oxidative phosphorylation, and the urea cycle are all conducted inside mitochondria [29]. Mitochondrial dysfunction leads to the excessive production of reactive oxygen species (ROS), and is the major source of ROS [30]. ROS can attack polyunsaturated fatty acids in membrane phospholipids, thereby producing large amounts of lipid peroxides (MDA), which is used as an index that reflects the oxidative status [31]. Additionally, SOD and GSH-PX, vital scavengers of ROS in the body [32], can inhibit lipid peroxidation. When hepatocytes are
NAFLD, which is closely associated with metabolic pathologies, is the most common chronic liver disease worldwide. Alarming epidemics of metabolic syndrome and obesity have accelerated the global prevalence of NAFLD [25]. Therefore, in the present study, an HFD-induced NAFLD model was established to study the alleviating effects of PK extract and its underlying mechanism of action, with RES as the positive control (because RES can effectively regulate lipid metabolism and mitochondrial function to alleviate NAFLD [26,27]). NAFLD is associated with an excessive lipid accumulation in the liver (which is beyond the scavenging ability of liver), and is mainly characterized by increases in TC, TG, and LDL-C content in the serum and/or liver, and a reduced HDL-C content [28]. We found that HFD-feeding resulted in notable increases in ALT, AST, TC, and LDL-C level in serum and TC and TG level in the liver. In addition, HFD-feeding results in reduced HDL-C
Fig. 4. Effects of HFD and PK on fatty acid metabolism. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/kg/day). After 14 weeks of HFD-feeding, rats were sacrificed, and the liver was collected. Expression levels of (A) CPT-1 mRNA and (B) UCP-2 mRNA were evaluated using real-time quantitative reverse transcription polymerase chain reaction. Values represent the mean ± S.D. from 7 animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group. CPT-1, Carnitine palmitoyltransferase-1; UCP2, uncoupling protein-2.
6
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Fig. 5. Effects of HFD and PK on hepatocellular apoptosis. SD rats were administered either saline, RES or PK extract (1, 2, or 4 g/ kg/day). After 14 weeks of HFD-feeding, rats were sacrificed, and the liver was collected. Hepatocellular mitochondria were isolated for determining the expression of cytchrome c. Expression levels of (B) caspase-9 (B1, 37 KD; B2, 50 KD), (C) caspase-3 (C1, 32 KD; C2, 17 KD), (D) Bcl-2, (E) Bax, (F) cytochrome c were evaluated by Western blot analyses. Immunoreactive protein bands (A) were visualized with a chemiluminescence horseradish peroxidase substrate. Values represent the mean ± S.D. from 6 randomly-selected animals. * P < 0.05, ** P < 0.01, *** P < 0.001 versus the relevant HFD group.
oxidation of short-chain (< C8), medium-chain (C8–C12), and longchain (C12–C20) fatty acids [34]. Short- and medium-chain fatty acids can freely enter the mitochondria, while long-chain fatty acids only enter the mitochondria with the assistance of CPT-1 transport [23]. Thus, mitochondrial CPT-1 is the primary rate-limiting enzyme of βoxidation. Increasing the activity or expression of CPT-1 will facilitate the oxidation of fatty acids accumulated in the liver, which is beneficial for alleviating NAFLD. We demonstrated that HFD-feeding significantly down-regulated the expression of CPT-1 mRNA in the liver, which was significantly inverted by treatment with PK extract, demonstrating that PK accelerated beta-oxidation of fatty acid within the liver to protect HFD-induced NAFLD rats. In previous studies, it was demonstrated that apoptosis aggravated hepatocellular injury. The release of cytochrome c is a critical step in the apoptotic process [35,36]. Cytochrome c is released into the cytoplasm and binds to apoptotic proteases, thereby activating factor-1 (Apaf-1) to form a polymer in the presence of deoxyadenosine triphosphate (dATP), which promotes its binding with caspase precursor protein (pro-caspase) to form apoptotic bodies and further activate caspase-9. Activated caspase-9 can activate other caspases (e.x. caspase3), which results in apoptosis. Bax, one of the earliest pro-apoptotic proteins discovered, mainly resides in the cytoplasm in normal cells. However, upon stimulation by apoptotic signals, it can be translocated to mitochondria, which causes the release of cytochrome c into the cytoplasm [37–39]. Bcl-2 is the main anti-apoptotic protein in its family of proteins and contains four domains (BH1 to BH4) that stabilize mitochondrial membrane function and prevents mitochondria releasing apoptotic factors, such as apoptosis-inducing factor and cytochrome c [39,40]. We found that HFD-feeding significantly increased the expression of caspase 9, caspase 3 and Bax in hepatocytes, and decreased the expression of Bcl-2 in hepatocytes and cytchrome c in mitochondria. These effects were significantly inverted by PK treatment, indicating that PK inhibited hepatocellular apoptosis and decreased liver injury to alleviate HFD-induced NAFLD. In addition, it is clearly noted that the effects of three doses of PK
excessively attacked by ROS, their levels may be decreased. Therefore, determining MDA, SOD, and GSH-PX levels not only reflect the degree of liver damage, but also reflect the effect of drugs on the oxidative stress status in hepatocytes. We found that HFD-feeding led to a significant increase in MDA and reduced SOD and GSH-PX level in liver mitochondria from NAFLD rats. However, these effects were significantly restored by PK treatment, suggesting that PK may enhance the body's ability to scavenge ROS from mitochondria and further alleviate its lipid peroxidation of cell membranes to exert a protective effect on NAFLD rats. ATP, as a major energy substance in the body, is mainly synthesized in mitochondria, and depends on the activity of complexes on the mitochondrial respiratory chain [29]. When the function of the respiratory chain is damaged, energy metabolism will be prevented. This effect will decrease the ability of hepatocytes to treat and transport liver lipids, which may result in lipid accumulation within the liver and further aggravate HFD-induced NAFLD. In addition, lipid accumulation in the liver will up-regulate UCP-2 expression and further decrease ATP level, which not only decreases energy supply of TC synthesis and further inhibits lipid formation, but also promotes the β-oxidation of fatty acids and further alleviates liver lipid accumulation [24]. However, excessive UCP-2 expression will decrease the ATP reserve in mitochondria [24,33], which aggravates liver injury when the ATP requirement of hepatocytes is significantly increased. In this study, it was found that the activities of ATPase, and complex I and II in the liver mitochondria were reduced by HFD-feeding, with the increasing of UCP-2 mRNA expression in liver, whereas the effects were inverted by treatment with PK supplement, indicating that PK may promote oxidation rate of liver respiratory chain and decrease oxidative phosphorylation uncoupling, which further enhances mitochondrial energy metabolism to alleviate HFD-induced NAFLD. Lipid metabolism disorder can increase the supply of fatty acids in the liver, resulting in the accumulation of fatty acids in the liver, leading to NAFLD. Mitochondrial β-oxidation is the main oxidative pathway for the disposal of fatty acids and primarily involves the 7
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Fig. 6. The regulation of PK extract on HFD-induced NAFLD.
Acknowledgments
extract on the assayed indicators did not show dose-dependent manners, which was similar to many other herbal medicines [41,42]. This may be attributed to the complex constituents and pharmacological mechanism of the herbs (e.g., a mode of action of multi-compound, multi-pathway and multi-target [43]). Besides, it is very likely that the PK dosages used in our study were not in the range of displaying dosedependence.
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5. Conclusion Taken together, our data indicated that PK alleviated HFD-induced NAFLD and multiple risk factors by promoting mitochondrial functions, which may be related to the enhancement of mitochondrial anti-oxidative status, energy metabolism and beta-oxidation of fatty acid, as well as inhibition of hepatocellular apoptosis (Fig. 6). PK may be used for developing mitochondrial regulators/nutrients to remedy NAFLDassociated mitochondrial dysfunction to further alleviate NAFLD. Further investigation regarding the effects of PK on the absorption and excretion of lipids remains of investigative interest. Author contributions Xing-Xin Yang and Xi Wang wrote the manuscript. Xi Wang, XingXin Yang, Jin-Cai Dong and Feng-Jiao Li conducted the experiments. Ting-Ting Shi provided technical support and helpful discussions. Lin-Xi Zeng, Min Yang, Wen Gu and Jing-Ping Li participated in writing and modifying the manuscript. Jie Yu and Xing-Xin Yang designed the study. All authors have no conflicts of interest. Ethics approval and consent to participate Approval from the Institutional Ethical Committee on Animal Care and Experimentations of Yunnan University of Chinese Medicine (R0620160026) was obtained for this study. Funding This study was supported by grants from the National Natural Science Foundation of China (Grants 81660596, 81460623 and 81760733), and the Application and Basis Research Project of Yunnan China (Grants 2016FD050 and 2017FF117-013). Availability of data and materials All data used to support the findings of this study are available from the corresponding author upon request. Consent for publication Publication of the manuscript has been approved by all co-authors. 8
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