Highly bioavailable berberine formulation ameliorates diabetic nephropathy through the inhibition of glomerular mesangial matrix expansion and the activation of autophagy

Journal Pre-proof Highly bioavailable berberine formulation ameliorates diabetic nephropathy through the inhibition of glomerular mesangial matrix expansion and the activation of autophagy Meishuang Zhang, Yining Zhang, Dong Xiao, Jing Zhang, Xinxin Wang, Fengying Guan, Ming Zhang, Li Chen PII:

S0014-2999(20)30047-9

DOI:

https://doi.org/10.1016/j.ejphar.2020.172955

Reference:

EJP 172955

To appear in:

European Journal of Pharmacology

Received Date: 6 November 2019 Revised Date:

17 January 2020

Accepted Date: 24 January 2020

Please cite this article as: Zhang, M., Zhang, Y., Xiao, D., Zhang, J., Wang, X., Guan, F., Zhang, M., Chen, L., Highly bioavailable berberine formulation ameliorates diabetic nephropathy through the inhibition of glomerular mesangial matrix expansion and the activation of autophagy, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2020.172955. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Highly bioavailable berberine formulation ameliorates diabetic nephropathy through the inhibition of glomerular mesangial matrix expansion and the activation of autophagy Meishuang Zhang 1, Yining Zhang2, Dong Xiao1, Jing Zhang1, Xinxin Wang3, Fengying Guan1, Ming Zhang 1*, Li Chen 1*

1.Department of Pharmacology, College of Basic Medical Sciences, School of nursing, Jilin University, Changchun 130021, China 2.Research Institution of Paediatrics, Department of Pediatric Endocrinology, The First Clinical Hospital Affiliated to Jilin University, Changchun 130021, China 3. Senior Officials Inpatient Ward, The First Clinical Hospital Affiliated to Jilin University, Changchun, 130021, China

✉Corresponding author: Dr. Li Chen, Professor and Director, Department of Pharmacology, College of Basic Medical Sciences, School of nursing, Jilin University, 126 Xin Min Street, Changchun, Jilin 130021, China. E-mail: [email protected], Tel: +86-0431-85619799 Dr. Ming Zhang, Associated Professor, Department of Pharmacology, College of Basic Medical Sciences, Jilin University, 126 Xin Min Street, Changchun, Jilin 130021, China. E-mail: [email protected], Tel: +86-0431-85619799

Abstract: Glomerular mesangial matrix expansion and cell autophagy are the most important factors in the development of kidney damage under diabetic conditions. The activation of AMPK might be an important treatment target for diabetic nephropathy. Berberine has multiple effects on all types of diabetic complications as an activator of AMPK. However, the poor bioavailability of berberine limits its clinical applications. Huang-Gui Solid Dispersion (HGSD), a new formulation of berberine developed in our lab, has 4-fold greater bioavailability than berberine. However, its therapeutic application and mechanism still need to be explored. In the present study, the effect of HGSD on kidney function in type 2 diabetic rats and db/db mice was investigated. The results demonstrated that HGSD improved kidney function in these two animal models, decreased the glomerular volume and increased autophagy. Meanwhile, AMPK phosphorylation levels and autophagy-related protein expression were significantly increased, and extracellular matrix protein deposition-related protein expression was decreased after treatment. The present study indicated that HGSD protected against diabetic kidney dysfunction by inhibiting glomerular mesangial matrix expansion and activating autophagy. The mechanism of HGSD in the treatment of diabetic nephropathy might be connected to the activation of AMPK phosphorylation. Keywords: Huang-Gui Solid Dispersion, Diabetic nephropathy, AMPK, Autophagy, Extracellular matrix deposition

1. Introduction Diabetic nephropathy (DN) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal failure. Recent evidence has suggested that the mechanism of diabetic nephropathy is associated with autophagy and extracellular matrix deposition. Autophagy is a fundamental cellular homeostatic process that degrades and recycles cellular proteins and removes damaged organelles (Liu et al., 2018). The classical mTOR pathway plays a major role in the negative regulation of autophagy (Li et al., 2015). The mTORC1 complex suppresses autophagy via the phosphorylation and inactivation of ULK1, an initiator of autophagosome formation (Puente et al., 2016). Clinically, mTORC1 activity is enhanced in DN patients with type I or type II diabetes (Godel et al., 2011). Research has confirmed that inhibition of mTOR can improve autophagy and improve kidney protection (Xiao et al., 2014). Furthermore, extracellular matrix (ECM) deposition is closely related to diabetic nephropathy, and ECM deposition is mainly due to an imbalance between the abnormal synthesis of various components and degradation, such as fibronectin(FN), collagen

(Col ), and collagen

(Col

)). ECM deposition is considered to be an important

mechanism that causes glomerulosclerosis (Ding et al., 2010). It has been confirmed that TGF-β1 and other cytokines can regulate ECM metabolism. The mechanism may involve the specific regulation of the synthesis and degradation of the ECM by TGF-β1, which can increase ECM synthesis and decrease degradation (Chen et al., 2005; Ziyadeh, 2004). The key gene correlated with these two effects might be a target for the treatment of diabetic nephropathy. 5’-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a major regulator of whole-body energy homeostasis in multiple key organs, such as the liver, skeletal muscle, and kidneys (Glosse and Foller, 2018; Pastor-Soler and Hallows, 2012; Rajani et al., 2017; Ramesh et al., 2016). It has been reported that AMPK regulates epithelial transport, epithelial-to-mesenchymal transition, and extracellular matrix (ECM) deposition in the kidney (Allouch and Munusamy, 2018; Jin et al., 2017; Sanchez et al., 2011; Tain and Hsu, 2018; Tsai et al., 2014). AMPK activation can inhibit the expression of TGF-β1 and proliferation of mesangial cells under high-glucose conditions (Sanchez et al., 2011). Meanwhile, AMPK can phosphorylate TSC1/2 to inhibit mTORC1 or directly phosphorylate ULK1, which functions

as a positive regulator of autophagy (Jin et al., 2017). Therefore, it can be predicted that AMPK might be the key therapeutic target for preventing the progression of DN via the promotion of autophagy, in inhibition of TGF-β1 expression, and a reduction in the deposition of the extracellular matrix. Berberine presents multiple pharmacological effects on diabetes and diabetic complications and has been identified as an agonist of AMPK (Al-Rasheed et al., 2015; Jin et al., 2017; Kitada et al., 2011; Tang et al., 2014). However, its effect on DN still needs to be further explored. Furthermore, the poor solubility and membrane permeability of berberine prohibit its clinical usage for the treatment of diabetes. Huang-Gui Solid Dispersion (HGSD), a new formulation of berberine developed in our lab, has been demonstrated to possess anti-diabetic activities and 4-fold greater bioavailability than berberine (Zhaojie et al., 2014). However, its therapeutic application and mechanism still need to be explored. The present study was designed to investigate the therapeutic effects of HGSD on diabetic nephropathy and elucidate its underlying mechanisms. High-fat plus multiple STZ injection-induced diabetic nephropathy model rats and genetically diabetic Leprdb/db mice were used. The protective effect of HGSD on DN was observed. Alterations in AMPK, TGF-β, ECM deposition and autophagy proteins were detected. 2. Materials and methods 2.1 Materials Ternary HGSD was prepared with berberine (purity quotient >99.8%) obtained from Northeast Pharmaceutical Group Co., Ltd. (Shenyang, China), sodium caprate and carriers (PVP K30, PEG6000 and F68) by solvent evaporation[21]. Sodium caprate was purchased from Sigma-Aldrich Co., LLC (St. Louis, USA). PVP K30, PEG6000 and F68 were purchased from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Other reagents were purchased from Beijing General Chemical Reagent Factory (Beijing, China). The following reagents were used: STZ (Sigma-Aldrich, USA), a glucose test kit, creatinine, urea nitrogen, urinary albumin and urine microalbumin kit (Jiancheng Technology Co., Nanjing, China), anti-p-AMPK (ab133448, Abcam, UK), anti-AMPK (ab32047, Abcam, UK), anti-mTOR (sc-8319, Santa Cruz Biotechnology, USA), anti-p-mTOR (sc-101738, Santa Cruz Biotechnology, USA), anti-ULK1 (#8054, Cell Signaling Technology, USA),

anti-p-ULK1 (#6887, Cell Signaling Technology, USA), anti-beclin-1 (sc-11427, Santa Cruz Biotechnology, USA), anti-LC3(Ι/ ) (A7198, ABclonal Technology, USA), anti-TGF-β1 (sc-52893, Santa Cruz Biotechnology, USA), anti-α-SMA (ab5694, Abcam, UK), and anti-GAPDH (sc-365062, Santa Cruz Biotechnology, USA) antibodies, an anti-mouse secondary antibody (SA00001-1, Proteintech Group, USA), and an anti-rabbit secondary antibody (SA00001-2, Proteintech Group, USA). 2.2 Experimental Animals The diabetic rat model was generated by our lab based on improvement to a previously described method (Penumathsa et al., 2008). Regular chow consisting of 5% fat, 53% carbohydrate, and 23% protein with a total calorific value of 25.0 kJ/kg and a high-fat diet consisting of 22% fat, 48% carbohydrate, and 20% protein with a total calorific value of 44.3 kJ/kg were obtained from the Experimental Animal Holding Facility of Jilin University. Ninety male Wistar rats (Experimental Animal Holding Facility of Jilin University) weighing 180 to 220 g were housed individually in cages in a temperature-controlled room with a 12h light:dark cycle. After 1 week of acclimation with free access to regular rodent chow and water, the rats were randomly divided into 2 groups. Group 1 (n=12, normal control [CON]) was fed regular chow. Group 2 (n=78, high-fat diet [HFD]) was fed the high-fat diet. After 4 weeks of high-fat diet feeding, group 2 was injected with STZ dissolved in citrate buffer (pH 4.5) at a dose of 30 mg/kg body weight and tested for fasting blood glucose (FBG) levels 4 weeks post-injection. Group 1 was injected with the citrate buffer vehicle. The rats in group 2 with an FBG level less than 7.8 mmol/l were injected with STZ again (30 mg/kg), while the rats in group 1 were given vehicle citrate buffer at a dose of 0.25 ml/kg IP. Four weeks after STZ injection, the rats with an FBG level ≥7.8 mmol/l measured twice or with a nonfasting FBG level ≥ 11.1 mmol/l were randomly divided into 4 groups and continued on the high-fat diet. One group was used as a high-fat diabetic nephropathy model (DM), and the other 2 were orally gavaged with HGSD dissolved in 0.5% CMC-Na at a dose of 25 (H25) or 100 (H100) mg/kg body weight per day. The other group was gavaged with berberine dissolved in 0.5% CMC-Na at a dose of 100 (BER) mg/kg body weight per day. Rats in the CON and DM groups were gavaged with 0.5% CMC-Na. After 16 weeks, the animals were fasted overnight, and urine was collected with metabolic cages. Blood samples obtained from the tails were

collected in EDTA tubes and placed on ice. After centrifugation, the plasma was collected and stored at -80°C. The kidneys were immediately separated, weighed, collected and stored in liquid nitrogen until further analysis. Thirty male BKS.Cg-Dock7m+/+Leprdb/JNju mice (db/db mice) and ten control mice (con) (4–8 weeks of age) were procured from Nanjing Biomedical Research Institute of Nanjing University. The mice were housed under standard laboratory conditions (23 ± 1°C, 40–60% relative humidity, and a 12h light-dark cycle) in the Experimental Animal Holding Facility of Jilin University. All mice were allowed free access to normal chow diet and tap water. After one week of acclimation, the db/db mice were randomly divided into three groups (n=10): the model group (model) and 2 other groups that were orally gavaged with HGSD dissolved in 0.5% CMC-Na at a dose of 40 (HG-L) or 160 (HG-H) mg/kg body weight per day. The mice in the con and model groups were gavaged with 0.5% CMC-Na. The treatment lasted for four weeks. At the end of the experiment, all animals were fasted overnight, and urine was collected with metabolic cages. Whole blood was collected, and the serum was isolated for biochemical analysis. All animal experimental procedures were approved by the Ethics Committee for the Use of Experimental Animals of Jilin University [SYXK(Ji)2013-0005]. 2.3 Glucose tolerance test and biochemical indicator detection At the end of the experimental period of 16 weeks (or 4 weeks for db/db mice), the rats or mice were housed in metabolic cages for over 24h for the collection of urine samples. Urine albumin, urine creatinine, urine BUN, plasma urea and creatinine concentrations, and FN, Col Ι and Col

levels were measured by ELISA.

2.4 Histopathology Rats and mice were anaesthetized under light ether anaesthesia and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 5 min to fix the tissues. Two-micrometer-thick cross-sections of the kidneys were prepared and stained with haematoxylin and eosin to evaluate renal pathology. After glomerular areas were measured using Image-Pro Plus software, version 6.0 (Media Cybernet-ics, USA), glomerular volumes were calculated using the Weibel and Gomez formula (glomerular volume =area 1.5×1.38÷1.01, where 1.38 indicates the shape coefficient and 1.01 indicates the size

distribution coefficient). 2.5 Transmission electron microscopy At the same time, pathological changes in kidney tissues were observed by transmission electron microscopy. Renal cortical tissues (1 mm3) were fixed in 2.5% glutaraldehyde to produce ultrathin sections. The ultrathin sections were observed by electron microscopy at a magnification of 10,000x. Two glomeruli were observed for each sample. Ten photos were taken for each glomerulus through continuous manual acquisition. Changes in the glomerular mesangial matrix were observed. 2.6 Western blot analysis Proteins were extracted from kidney tissues following the manufacturer's protocols (Beyotime, China). The protein concentration was quantified using the BCA protein assay kit (Thermo Fisher Scientific, USA), and 30 µg protein was separated on a 12% SDS-poly-acrylamide denaturing gel and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). The membranes were blocked with 5% (w/v) BSA for 2h at room temperature and then incubated with primary antibodies with light shaking overnight at 4°C. Primary antibodies against phospho-AMPK, AMPK, phospho-mTOR, mTOR, phospho-ULK1, ULK1, beclin-1, LC3(Ι/ ), TGF-β1, α-SMA and GAPDH were diluted 1:1000 in TBST buffer. The membranes were washed 3 times for 5 min each with 10 ml TBST (10 mM Tris-HCl, 150 mM NaCl and 0.1% (v/v) Tween 20) and then incubated with secondary antibody at room temperature for 2h. Goat anti-rabbit and goat anti-mouse secondary antibodies (Proteintech, USA) were diluted 1:5000 in TBST buffer. The membranes were incubated in Western ECL substrate (Thermo Fisher or Proteintech, USA) and exposed with Tanon Imager software using Quantity One software for image analyses. 2.7 Statistical analysis The data are presented as the mean ± S.E.M.. The statistical significance of the differences was analysed by one-way analysis of variance (ANOVA). All statistics were calculated with SPSS 13.0 software (SPSS Inc., USA). The significance level for all comparisons was P <0.05.

3. Results 3.1 Diabetic kidney function was improved by HGSD After 16 weeks of the experiment, fasting blood glucose (FBG) levels, the area under the curve (AUC) of the IPGTT results, kidney/body weight, and serum creatinine and blood urea nitrogen levels were detected in the rats. The FBG levels and AUCs of DM rats were approximately 3.89 times and 2.69 times higher, respectively than those of CON rats. The FBG bar graphs show that the FBG levels were 1.93 and 1.32 times higher in the H25 and H100 groups, respectively, and 2.86 times higher in the BER group than in the CON group. The AUCs of the IPGTT results were 1.91 and 1.69 times in the H25 and H100 groups, respectively, and 2.43 times higher in the BER group than in the CON group. At the same time, kidney/body weight and serum creatinine and blood urea nitrogen levels in the DM group were approximately 2.03, 4.36 and 2.06 times higher than those in the CON group. Kidney/body weight in the H25, H100 and BER groups of diabetic rats were 0.40, 0.45 and 0.46 times lower than that in the DM group. The serum creatinine levels of DM rats treated with 25 mg/kg, 100 mg/kg HGSD or 100 mg/kg BER were approximately 0.32, 0.20 and 0.24 times lower, respectively, and blood urea nitrogen levels of these rats were approximately 0.58, 0.50 and 0.77 times lower, respectively, than those in the DM group (Fig. 1). Furthermore, after 4 weeks of the experiment, fasting blood glucose (FBG) levels, the area under curve (AUC) of the OGTT results, kidney/body weight, and blood creatinine and blood urea nitrogen levels were detected in the db/db mice. The FBG levels and AUCs in the model group were approximately 3.25 times and 3.86 times higher than those in the con group. The FBG levels of model mice treated with 40 mg/kg or 160 mg/kg HGSD were approximately 0.67 and 0.66 times lower, respectively, and the AUCs of these mice were 0.66 and 0.60 times lower, respectively, than those of the model group. Meanwhile, the blood creatinine and blood urea nitrogen levels in the model group were approximately 1.17 and 1.16 higher than those in the con group. The blood creatinine levels of model mice treated with 40 mg/kg or 160 mg/kg HGSD were approximately 0.55 and 0.58 times lower, respectively, and the blood urea nitrogen levels of these mice were approximately 0.73 and 0.57 times lower, respectively, than those of the model group, but kidney/body weight was not

changed compared with that of the model group (Fig. 2). Additionally, we detected 24h urine protein excretion and 24h urine microalbumin, urine creatinine and urine BUN levels in rats. The 24h urine protein excretion and 24h urine microalbumin levels in the DM group were approximately 2.68 and 1.45 times higher than those in the CON group, and the urine creatinine and urine BUN levels were approximately 0.27 and 0.58 times lower in the DM group than in the CON group. Twenty-four-hour urine protein excretion was 0.23 and 0.48 times lower in the H25 and H100 groups and 0.48 times lower in the BER group than in the DM group. Furthermore, 24h urine microalbumin levels were 0.66, 0.63 and 0.69 times lower in the H25, H100 and BER groups, respectively, than in with the DM group. Urine creatinine levels were 2.08 and 4.18 times higher in the H25 and H100 groups, respectively, and 1.85 times higher in the BER group than in the DM group. Additionally, urine BUN levels were 1.63 and 1.72 times higher in the H25 and H100 groups, respectively, and 1.32 times in the BER group than in the DM group (Fig. 3). Consistent with the above results, 24h urine protein excretion, 24h urine volume and urine BUN levels in the model group were approximately 3.24, 6.29 and 2.27 times higher than those in the con group. Urine creatinine levels were approximately 0.25 times lower in the model group than in the con group. In addition, 24h urine protein excretion was 0.56 and 0.14 times lower in the HG-L and HG-H groups, respectively, than in the model group. The 24h urine volume levels were 0.34 and 0.15 times lower in the HG-L and HG-H groups than in the model group. The urine creatinine level was 1.57 and 2.07 times higher in the HG-L and HG-H groups, respectively, than in the model group, but the urine BUN level was not changed compared with that in the model group (Fig. 4). 3.2 HGSD improved renal morphological changes The light microscopic observations of the from in the control group of rats and db/db mice showed normal glomeruli (Fig. 3E, Fig. 4E). In contrast, the kidneys of diabetic rats and model mice showed marked histological changes in the cortex, indicating an increased number of mesangial cells in areas of expanded mesangium. Meanwhile, mesangial matrix expansion and mesangial cell hyperplasia were attenuated by HGSD treatment in both animal models. The changes in the mean glomerular volume in the experimental groups are illustrated (Fig. 3F, Fig. 4F). The mean glomerular volumes of the untreated DM group and

the model group were increased compared to those of the controls (P <0.01). Notably, this increase was partially recovered following HGSD administration. These observations indicated that HGSD treatment can prevent renal structural changes in diabetes in experimental animals. The electron microscopy data suggested changes in the glomerular mesangial and autophagy levels. Compared with those in the con group, the mesangial cells in the model group proliferated significantly. The mesangial cells in the HGSD groups showed some recovery compared with those in the model group. These results suggested that HGSD can effectively protect kidney tissue and reduce mesangial cell proliferation. Compared with those in the con group, podocytes in the model group exhibited fused, absent, or irregular nuclei. Rough endoplasmic reticulum cavity expansion was observed, most mitochondria exhibited vacuoles. The golgi complex was also found in the cytoplasm, but few autophagosomes were observed. Fewer fused podocytes were observed in the HGSD group than in the model group. The endoplasmic reticulum and mitochondrial structure were not changed. In particular, the number of podocytes in the 160 mg/kg HGSD group was greater than that in the 40 mg/kg HGSD group. Based on these results, HGSD effectively protected the podocyte structure and increased the number of autophagosomes (Fig. 5A). Consistent with these observations, the levels of FN and Col Ι were also increased in the model group compared to the db/db mouse con group. The FN levels of model mice treated with 40 mg/kg or 160 mg/kg HGSD were approximately 0.59 and 0.53 times lower, respectively, and the Col Ι levels of these mice were approximately 0.60 and 0.53 times lower, respectively, than those of the model group (Fig. 5B/C). The level induced by HGSD treatment was comparable to that observed in untreated non-diabetic animals, indicating that HGSD had a protective effect against DN. 3.3 The effect of HGSD on autophagy-related indicators in the kidneys It has been reported that mTOR plays an important role in metabolism and inhibits autophagy. AMPK can promote p-ULK1 (Ser317) expression and autophagy (Jung et al., 2009; Long et al., 2016). Western blot analysis showed that the p-AMPK/AMPK and p-ULK1/ULK1 levels in the DM group were approximately 0.61 and 0.09 times lower, respectively, and that the p-mTOR/mTOR level was 3.14 times higher than those in the CON group. In contrast, diabetic rats treated with 25 mg/kg HGSD or 100 mg/kg HGSD showed a

robust increase in p-AMPK/AMPK expression (approximately 2.11 and 2.93 times higher, respectively) and an increase in the P-ULK/ULK level (3.56 and 19.22 times higher, respectively) compared with those in untreated DM rats. The p-mTOR/mTOR level were 0.67 and 0.38 times lower than that in DM rats (Fig. 6 A/B/C/D). The expression level of LC3- /LC3-

is related to the number of autophagosomes,

while beclin-1 promotes the maturation of autophagosomes. The data showed that, compared with those in the con group, the LC3- /LC3-

and beclin-1 levels in the DM group were

0.46 and 0.47 times lower, respectively. In contrast, diabetic rats treated with 25 mg/kg HGSD and 100 mg/kg HGSD exhibited LC3-II/LC3-I levels that were approximately 3.30 and 6.67 times higher, respectively and beclin-1 levels that were 6.83 and 9.28 times higher, respectively, than those in DM rats (Fig. 6 E/F/G). 3.4 The effect of HGSD on ECM deposition-related indicators in the kidneys Compared with those in the con group, the FN, Col Ι and Col

levels in the DM groups

were significantly increased in diabetic rats. The FN level of DM rats treated with 25 mg/kg or 100 mg/kg HGSD was approximately 0.81 and 0.85 times lower, respectively, the Col Ι level of these rats was approximately 0.89 and 0.80 times lower, respectively, and the Col level of these rats was by 0.91 and 0.77 times lower than the levels of the DM group (Fig. 7A/B/C). We further investigated the expression levels of TGF-β1 and α-SMA. TGF-β1 can promote the deposition of ECM and make the glomerulus gradually harden, while α-SMA is a characteristic protein synthesized after the transdifferentiation of renal intrinsic cells into MyoF, which can indirectly reflect the degree of renal fibrosis. These data suggested that the DM group exhibited protein expression levels of TGF-β1 and α-SMA in the kidneys that were approximately 2.13 and 2.22 times higher, respectively, than those in the CON group. The expression of TGF-β1 was 0.72 and 0.62 times lower in the H25 and H100 groups, respectively, than in the DM group. The expression of α-SMA was 0.55 and 0.27 times lower in the H25 and H100 groups, respectively. The results showed that TGF-β1 and α-SMA were activated in the kidneys of diabetic animals and that HGSD inhibited the expression levels of TGF-β1 and α-SMA (Fig. 7D/E/F).

4. Discussion Increasing evidence has suggested that diabetic nephropathy is induced by multiple factors, such as dyslipidaemia, hyperglycaemia, haemodynamic abnormalities and oxidative stress (Gilbert and Cooper, 1999; Najafian et al., 2011; Ziyadeh, 2004). Studies have shown that berberine can treated diabetic nephropathy (Li and Zhang, 2017; Zhu et al., 2018), but the mechanism is still unclear, and its poor bioavailability limits its clinical applications. At the same time, studies have shown that autophagy and extracellular matrix deposition are closely related to the development of diabetic nephropathy (Ebrahim et al., 2018; Feng et al., 2018; Zhou et al., 2018). We previously demonstrated that HGSD, developed in our laboratory, can improve brain and myocardial ischaemia-reperfusion injury in rats and attenuate neuronal apoptosis by enhancing autophagy in the brain of diabetic mice with a remarkable hypoglycaemic effect (Chen et al., 2016; Xue et al., 2016; Yu et al., 2018; Zhaojie et al., 2014). In the current study, we demonstrated that HGSD plays an important role in ameliorating DN. The principal findings of our study are as follows: 1. as a highly bioavailable berberine formulation, HGSD ameliorates diabetic nephropathy; and 2. this effect is related to the activation of autophagy and the inhibition of extracellular matrix deposition via AMPK activation. Berberine is a Chinese traditional medicine that has long been used to treat diarrhoea. Previous studies have shown that berberine has an ameliorative effect on diabetes complications, such as cardiac dysfunction and endothelial dysfunction in diabetic rats (Chang et al., 2013; Chang et al., 2012), but its low solubility and poor membrane permeability has limited its application in the clinic for the treatment of diabetes and its complications. Our laboratory developed an amorphous solid dispersion of berberine with absorption enhancer sodium caprate (SC), referred to as Huang-Gui Solid Dispersion (HGSD), and examined it for improved dissolution and oral bioavailability. The new formulation of berberine could simultaneously resolve the problem of the poor solubility and membrane permeability of berberine, which has been validated (Zhaojie et al., 2014). The present study demonstrated that HGSD exerts prominent effects on renal function in DN model rats and db/db mice. After administration of HGSD, fasting blood glucose levels, insulin sensitivity,

and serum creatinine, urea nitrogen, urine creatinine, urine urea nitrogen, 24h urine protein levels in diabetic rats and db/db mice were significantly improved. As suggested by pathological sections and the electron microscopy results, HGSD inhibited glomerular mesangial matrix expansion, increased the number of autophagosomes, and improved organelle micromorphology. The effect of treatment with 100 mg/kg berberine was similar to that of treatment with 25 mg/kg HGSD in DN model rats, which suggests improved bioavailability. However, the mechanism of the protective effect of HGSD against DN remains unclear. Autophagy is a regulatory lysosomal protein degradation pathway that can remove damaged organelles to maintain cellular homeostasis. The function of autophagy in the kidneys is currently under investigation, and it has been shown to have a renoprotective effect in acute kidney injury and diabetic nephropathy (Ding and Choi, 2015; Kaushal and Shah, 2016; Kume et al., 2012; Yasuda-Yamahara et al., 2015). There are correlations between AMPK and mTORC1 signalling during multiple steps of autophagy regulation. The activity of autophagy may be induced by AMPK, which inhibits mTORC1 activity via the phosphorylation of its regulatory-associated proteins. Recent studies have highlighted the importance of the AMPK-dependent phosphorylation of ULK1 in inducing autophagy. A balance between mTORC1 and AMPK may directly regulate ULK1 activity and subsequently initiate autophagy (Lee et al., 2010; Roach, 2011). Some previous studies in our laboratory have demonstrated that berberine is an agonist of AMPK and may have therapeutic potential to protect Müller cells from high-glucose-induced apoptosis by enhancing autophagy and activating the AMPK/mTOR signalling pathway (Chen et al., 2018; Hang et al., 2018). Meanwhile, studies have indicated that berberine enhances autophagy and protects against high glucose-induced injury in podocytes by promoting AMPK activation (Jin et al., 2017). We have verified this pathway at the cellular level, but it is not clear what its overall level is. In this study, we detected AMPK and autophagy-related proteins in the kidneys of diabetic rats treated with HGSD. The results showed that HGSD significantly reduced the expression of p-mTOR and increased the expression of p-AMPK and the autophagy-related proteins p-ULK1, LC3- /LC3-

and beclin-1. This suggests that HGSD may act to protect the

kidneys to treat diabetic nephropathy by activating AMPK and promoting autophagy.

Simultaneously, We noticed that studies have shown chronic or overactive autophagy can have severely detrimental effects in the heart, our results show the level of Beclin 1 and LC3- /LC3-

were 3 times higher than the controls with the lowest dose H25. But in our

study, the kidney in model group did not show overactivated autophagy, instead, the autophagy level is suppressed. Second, the application of HGSD enhanced the autophagy level of the models significantly, and it was observed from the functional indicators that HGSD not only does not affect the function of the kidney, but can improve kidney dysfunction caused by diabetes. Unlike simple cell experiments, studies of the disease itself can highlight the nature of the disease. Studies have shown that extracellular matrix deposition-induced renal fibrosis, in addition to autophagy, is a main cause of diabetic nephropathy (Zhou et al., 2018). An increasing number of studies have suggested that TGF-β1 is closely related to the occurrence and development of diabetic nephropathy. TGF-β1 activates plasminogen activator inhibitor-1 (PAI-1), while PAI-1 inhibits plasminogen activator, reduces plasmin, and reduces ECM degradation (Hubacek et al., 2006; Wu et al., 2012). Studies have shown that resveratrol can activate AMPK and inhibit the expression of collagen to protect against STZ-induced early diabetic renal nephropathy (Ding et al., 2010). Additionally, resveratrol can reduce the expression of TGF-β1 by inhibiting the TGF-β/Smad pathway, and the ERK1/2 pathway further inhibits the expression of Col IV and protects type 2 diabetic nephropathy (Chen et al., 2011). Studies have shown that berberine inhibits kidney hypertrophy by inhibiting the synthesis of p38 MAPK and TGF-β and inhibiting the accumulation of fibronectin (FN) and type IV collagen (Lan et al., 2012). However, the current mechanism of the treatment of diabetic nephropathy by berberine through the activation AMPK and the inhibition of the accumulation of the extracellular matrix has not been reported. Our results support the above mechanisms. After treatment with HGSD, significant improvements in FN, Col I and Col IV levels were observed in DN rats and db/db mice. The protein expression levels of TGF-β1 and α-SMA in the kidneys of the model rats group were significantly decreased, indicating that HGSD inhibited extracellular matrix deposition and blocked the process of renal fibrosis to treat diabetic nephropathy. Taken together, our data provide direct evidence that HGSD can effectively improve

diabetic nephropathy by promoting autophagy and inhibiting glomerular mesangial matrix expansion, suggesting that HGSD may be a promising candidate for the treatment of DN. Acknowledgments This work was supported by funding from the science and technology development projects of Jilin Province (20180201025YY, 20190201144JC, 20190103098JH), and Norman Bethune Program of Jilin University (2015224). Conflict of Interest Statement The authors declare that they have no conflict of interest. Author Contributions ZMS, CL and ZM have made contributions to conception and design; ZMS, XD and ZJ collected data; ZMS, ZYN and WXX analyzed and interpreted data; ZMS and GFY have been involved in drafting the manuscript and revising it critically for important intellectual content.

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Figure legends Fig. 1: HGSD improved renal function in diabetic rats. A diabetic rat model was established with STZ. The level of fasting blood glucose (A). The IPGTT (B) and the AUC (C). Kidney/body weight (D). Serum creatinine (E) and blood urea nitrogen (F) levels were measured by a biochemical kit. The data represent means ± S.E.M., n=8, **

P < 0.01 vs. the con group; #P < 0.05 vs. the DM group; ##P < 0.01 vs. the DM

group. Fig. 2: HGSD improved renal function in db/db mice. The level of fasting blood glucose (A). The OGTT (B) and the AUC (C). Kidney/body weight (D). Blood CRE (E) and blood BUN (F) levels were measured by a biochemical kit. The data represent the means ± S.E.M., n=10, *P < 0.05 vs. the con group; ***P < 0.001 vs. the con group; ##

P < 0.01 vs. the model group; ###P < 0.001 vs. the model group.

Fig. 3: Renal morphology of rats with diabetic nephropathy was improved by HGSD. A diabetic nephropathy rat model was established with STZ. The levels of 24h urine protein (A), 24h urine microalbumin (B), urine creatinine (C) and urea nitrogen (D). Glomeruli sections (E) and kidney sections stained with haematoxylin and eosin. 400×. Histopathological changes in kidney structure were assessed in at least 20 randomly selected tissue sections from each group studied at 400× magnification, and representative images of normal glomeruli morphology from a con animal are shown. STZ-induced DM rats showed glomerular hypertrophy prior to HGSD treatment. The treatment of diabetic rats with HGSD (H25 and H100) for 16 weeks resulted in a lesser extent of glomerular hypertrophy. Changes in the glomerular volume (F). The glomerular volumes of HGSD-treated diabetic rats were reduced in comparison with those of untreated diabetic rats. All data are shown as the means ± S.E.M., n=8, **P < 0.01 vs. the con group; #P < 0.05 vs. the DM group; ##P < 0.01 vs. the DM group. Fig. 4: Renal morphology of db/db mice was improved by HGSD. The levels of 24h urine protein (A), 24h urine volume (B), urine CRE (C) and urine BUN (D). Glomerular sections of db/db mice (E) and kidney sections stained with haematoxylin and eosin. 400×. Histopathological changes in kidney structure were assessed in at

least 20 randomly selected tissue sections from each group at 400× magnification, and representative images of normal glomerular morphology of a con mouse are shown. Model group mice showed glomerular hypertrophy prior to HGSD treatment. The treatment of model group mice with 40 mg/kg or 160 mg/kg HGSD for 4 weeks resulted in a lesser extent of glomerular hypertrophy. Changes in the glomerular volume (F). The glomerular volumes of HGSD-treated db/db mice were reduced in comparison with those of untreated diabetic mice. All data are shown as the means ± S.E.M., n=10, **P < 0.01 vs. the con group; ***P < 0.001 vs. the con group; #P < 0.05 vs. the model group; ##P < 0.01 vs. the model group; ###P < 0.001 vs. the model group. Fig. 5: Transmission electron microscopy of and ECM deposition-related indicators in db/db mice. Histopathological changes in renal tissue under electron microscopy among the four groups (the con (control) group), model group, HG-L group (HGSD 40 mg/kg), and HG-H group (HGSD 160 mg/kg)). Col Ι (B) and FN (C) levels were measured by a biochemical kit. The data in B and C represent the means ± S.E.M., n=10, *P < 0.05 vs. the con group; #P < 0.05 vs. the model group; ##P < 0.01 vs. the model group. Fig. 6: The effect of HGSD on autophagy-related indicators in the kidneys. The relative protein expression of p-mTOR, mTOR, p-AMPK, AMPK, p-ULK1 and ULK1 (A-D). The relative protein expression of beclin-1, LC3-I and LC3-II (E-G) in the rat kidneys. The data represent the means ± S.E.M., n=8, *P < 0.05 vs. the con group; **P < 0.01 vs. the con group; #P < 0.05 vs. the DM group; ##P < 0.01 vs. the DM group. Fig. 7: The effect of HGSD on ECM deposition-related indicators in the kidneys. A diabetic nephropathy rat model was established with STZ. Col Ι (A), FN (B) and Col

(C) levels were measured by a biochemical kit. The relative protein expression

of TGF-β1 and α-SMA (D-F) in the rat kidneys. The data represent the means ± S.E.M., n=8, *P < 0.05 vs. the con group; **P < 0.01 vs. the con group; #P < 0.05 vs. the DM group; ##P < 0.01 vs. the DM group.

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