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Protective effects of Weipixiao decoction against MNNG-induced gastric precancerous lesions in rats
Biomedicine & Pharmacotherapy 120 (2019) 109427
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Protective effects of Weipixiao decoction against MNNG-induced gastric precancerous lesions in rats
T
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Tiantian Caia,d, Chengzhe Zhanga,b,c, Xiaohui Zengb,c, Ziming Zhaob,c, , Yan Yana, Xuhua Yud,e, ⁎⁎⁎ Lei Wud,e, Lin Lind,e, , Huafeng Pana,⁎ a
Guangzhou University of Chinese Medicine, Guangzhou, Guanghdong, 510000, China Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine, Guangzhou, Guanghdong, 510000, China c Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, Guanghdong, 510000, China d Department of Respiratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou,Guanghdong, 510000, China e Department of Respiratory Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou,Guanghdong, 510000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Precancerous gastric lesions Glycolysis Weipixiao Rats
Gastric cancer is recognized as one of the most common cancer. In-depth research of gastric precancerous lesions (GPL) plays an important role in preventing the occurrence of gastric cancer. Meanwhile, traditional treatment provides a novel sight in the prevention of occurrence and development of gastric cancer. The current study was designed to assess the effects of therapy with Weipixiao (WPX) decoction on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced GPL rats and the underlying molecular mechanisms. After 10-weeks treatment, all rats were sacrificed. Histopathological changes of gastric tissue were assessed via hematoxylin-eosin (HE) and High-iron diamine-Alcian blue-Periodic acid-Schiff (HID-AB-PAS) staining. To be fully evidenced, RT-qPCR, Western blot and immunohistochemistry were used to detect the expressions of LDHA, CD147, HIF-1α, MCT4, PI3K, AKT, mTOR and miRNA-34a, which were crucial factors for evaluating GPL in the aspect of glycolysis pathogenesis. According to the results of HE and HID-AB-PAS staining, it could be confirmed that MNNG-induced GPL rats were obviously reversed by WPX decoction. Additionally, the increased gene levels of LDHA, CD147, MCT4, PI3K, AKT, mTOR and HIF-1α in model group were down-regulated by WPX decoction, while miRNA-34a expression was decreased and up-regulated by WPX decoction. The significantly increased protein levels of LDHA, CD147, MCT4, PI3K, AKT, mTOR and HIF-1α induced by MNNG were attenuated in rats treated with WPX decoction. In brief, the findings of this study imply that abnormal glycolysis in MNNG-induced GPL rats was relieved by WPX decoction via regulation of the expressions of LDHA, CD147, HIF-1α, MCT4, PI3K, AKT, mTOR and miRNA-34a.
1. Introduction Gastric cancer is recognized as one of the most common cancer [1]. China is one of the highest incidence regions of gastric cancer in the world [2] and the pathogenicity mechanism remains unclear. To date, surgical interventions, the primary choice for treatment of gastric carcinoma, are therapeutic in less than 40% of cases as gastric carcinoma patients are typically diagnosed at terminal stages [3]. Therefore, methods for early prevention and diagnosis of gastric cancer are required. The formation of gastric cancer contains consecutive steps, in which intestinal metaplasia and dysplasia are identified as gastric
precancerous lesions (GPL) [4]. In-depth research of GPL can provide a novel sight in preventing the occurrence of gastric carcinoma. Emerging researches have demonstrated that aerobic glycolysis which means cancer cells will produce a large amount of lactate regardless of the availability of oxygen is involved in gastric cancer [5–7]. There is a high possibility that abnormal glycolysis occurs in GPL and N-methylN'-nitro-N-nitrosoguanidine (MNNG) was chosen to induce GPL [8]. Thus, it is a novel point to further demonstrate whether abnormal glycolysis is involved in GPL. Glycolysis has been recognized as one of the hallmarks of cancers, thus we hypothesize that abnormal glycolysis occurs in GPL. Lactate
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Corresponding author at: 232 East Ring Road, Guangzhou University Town, Panyu District, Guangzhou, 510006, China. Corresponding author at: 111 Dade Road, Yuexiu District, Guangzhou, 510120, China. ⁎⁎⁎ Corresponding author at: Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine, Guangzhou, Guanghdong 510000, China. E-mail addresses: [email protected] (L. Lin), [email protected] (H. Pan). ⁎⁎
https://doi.org/10.1016/j.biopha.2019.109427 Received 16 June 2019; Received in revised form 29 August 2019; Accepted 2 September 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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Table 1 The components of WeiPiXiao. Plant Latin name
Family
Local name
English name
Used part
A. membranaceus(Fisch.)Bge. Pseeudostellaria heterophylla(Miq.)Pax ex pax et Hoffm Atractylodes macrocephala Koidz. Salvia miltiorrhiza Oldenlandia
Leguminosae Caryophyllaceae compositae Labiatae Rubiaceae
Huang Qi Tai Zi Shen Bai Zhu Dan Shen Bai Hua She She Cao
Astragalus Radix Pseudostellariae Atractylodes macrocephala Salvia miltiorrhiza Bge. Oldenlandia diffusa(Willd.)Roxb.
Root Root Root Root Grass
previously [26,27]. 50 Rats intake of MNNG were used as model group and 10 rats were randomly divided into normal group which were administered routinely. At the 16th week the model group (n = 40), including model group, positive(WFC, 0.2 g/kg), high dose WPX group (SD, WPX 19.8 g/kg/d, n = 10) and low dose WPX group (SD, WPX 9.9 g/kg/d, n = 10). After treatment for 10 weeks, all rats were humanely euthanized with sodium pentobarbital (140 mg/kg i.p.) after 12 h fasting, and the stomachs were removed immediately.
dehydrogenase A (LDHA), an enzyme which catalyses the last step of glycolysis, has been presented to be overexpressed in various types of cancers and tumor cells [9–12]. Overexpressed LDHA was regarded to promote progression of gastric cancer [5]. Overproduction and intracellular accumulation of lactate which leads to tumor cells death can be exported by coexpression of monocarboxylate transporters 4(MCT4) and CD147 [13,14]. Additionally, the tendency of LDHA and MCT4 expression is mediated by hypoxia inducible factor-1α (HIF-1α) [15,16]. Therefore, it is possible that inhibiting LDHA expression may be a vital point in the prevention of GPL. PI3K-Akt-mTOR signaling pathway is involved in the regulation of aerobic glycolysis which is crucial in tumor occurrence and development. In this signaling pathway, the activation of PI3K can induce the expression of Akt which is the regulate mTOR level. Previous studies have indicated that mTOR can regulate HIF-1α to mediate cellular metabolism [17,18]. Also of note, miRNA-34, a tumour suppressor, is capable of mediating the expression of LDHA and PI3K/Akt signaling pathway [19–21]. Weipixiao(WPX) decoction is a traditional herbal formula as shown in Table 1, contained Astragalus, Radix Pseudostellariae, Atractylodes macrocephala, Salvia miltiorrhiza Bge, Oldenlandia diffusa(Willd.) Roxb. On the theory of traditional Chinese medicine, it is believed that WPX can reverse GPL via strengthening spleen, dispersing blood stasis and detoxicating. In a clinical study, it was established that WPX may ameliorate the symptoms of CAG patients, demonstrating that WPX had a clear therapeutic effect in protect the gastric mucosa [22,23]. In addition, our previous studies demonstrated that WPX protected the gastric mucosa of rats from exposure to MNNG by regulating Wnt Pathway-Associated Proteins [24]. However, the further mechanisms responsible for the curative effects of WPX on GPL remain to be explored. Therefore, in the present research, we aim to determine whether WPX reverse GPL via inhibiting glycolysis process.
2.3. Histological staining All the rats were sacrificed and the stomach tissue was collected and fixed in 10% neutral-buffered formalin over 12 h. Then the samples were dehydrated in alcohol and xylene respectively. After dehydration, the samples were embedded in paraffin. Serial sections (4–5 μm thick) were obtained from the stomach and stained with hematoxylin and eosin (H&E) and High-iron diamine-Alcian blue-Periodic acid-Schiff (HID-AB-PAS) staining using standard techniques to observe the histological changes of the gastric mucosa and the types of intestinal metaplasia. 2.4. Reverse transcription‑polymerase chain reaction (RT‑qPCR) Total RNA was isolated from the gastric tissue samples using TRIzol reagent (BEIJING DINGGUO CHANGSHENG BIOTECHNOLOGY CO, LTD). RNA(2 μg) was reverse transcribed into Complementary DNA (cDNA) using the iQ5 Multicolor Real-Time PCR Detection system(BIORAD, USA). Subsequently, Real-time PCR analysis was carried out via iQ5 Multicolor Real-Time PCR Detection system(BIO-RAD, USA). And the DNA bands were standardization to those of GAPDH. The primer pairs were as follows: GAPDH forward, 5'-TCTCTGCTCCTCCCTGTTCT3' and reverse, 5'-TACGGCCAAATCCGTTCACA-3'; LDH-A forward, 5'TTGTTGGGGTTGGTGC-3' and reverse, 5'-TCCCTCTTGCTGACGG-3'; MCT4 forward, 5'-CCAGGCCCACGGCAGGTTTC-3' and reverse, 5'-GCC ACCGTAGTCACTGGCCG-3'; CD147 forward, 5'-GGCACCATCGTAACC TCTGT-3' and reverse, 5'-TCTTTCCCACCTTGATCCTG-3'; HIF-1α forward, 5'-CCCCTACTATGTCGCTTTCTTG-3' and reverse, 5'-AGGTTTGT GCTGCCTTGTATG-3'; PI3K forward, 5'-ACTAACTGGACCCTTGGATGC CTAC-3' and reverse, 5'-CAGCCAAGAACTATTGGCAAC-TGA-3'; AKT forward, 5'-TAGGCATCCCTTCCTTACAGC-3' and reverse, 5'-CGCTCAC GAGACAGGTGGA-3'.
2. Materials and methods 2.1. Preparation and qualitative analysis of WPX WPX were provided by the First Affiliated Hospital of Guangzhou University of Chinese Medicine, it is composed of five Chinese herbal medicines. The components and dosage of WPX is described in the Table 1. The qualitative analysis of WPX had been reaffirmed by highperformance liquid chromatography (HPLC) [25]. Specific chromatograms of the standard substances and the WPX are also displayed in the supplemental materials. Positive drug Weifuchun(WFC, 0.2 g/kg) was purchased from Hangzhou hu qingyutang pharmaceutical co. LTD (Lot: 1606663).
2.5. Western Blot analysis Radioimmunoprecipitation assay (RIPA) lysis buffer(500 μl) contained protease inhibitors(5 μl) was used to extracted total protein from the frozen tissue(500 μg), and the protein concentration was determined by the bicinchoninic acid assay (BCA). Proteins were separated by SDS-PAGE and transferred into nitrocellulose membranes. Membranes were probed overnight with respective antibodies [β-actin (1:1000 dilution, sigma, CST124E); LDHA(1:1000 dilution, proteintech, 21799-1-AP); MCT4(1:1500 dilution, Santa cruz, sc-376140); CD1147(1:2000 dilution, abcam, ab108317); HIF-1α(1:1500 dilution, abcam, ab2185); PI3k(1:2000 dilution, abcam, ab151549); AKT(1:1000 dilution, abcam, ab8805); mTOR(1:2000 dilution, abcam, ab32028)]. Subsequently, goat anti-rabbit or goat anti-mouse horseradish
2.2. Materials and animals A total of 60 male Sprague-Dawley (SD) rats aged 4–6 weeks, 180–200 g of weight were obtained from Guangdong Medical Laboratory Animal Center(44007200027077). The entire study was performed with the permission and supervision of the Animal Ethics Committee of Guangdong Province Engineering Technology Research Institute of T.C.M.(GDPITCM160129). Mild GPL-like gastric disease was induced in SD rats by exposing to combination of MNNG solution for 200 mg/L and feeding every other day for 26 weeks as described 2
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cell occurred in 12th week. Meanwhile, the incidence of dysplasia was slightly higher in 16th week than 8th and 12th week. Fig.1B showed the histopathological changes of the gastric mucosa. By contrast to control group, it could be observed that the thickness of basement membrane were more asymmetrical in model group. In addition, dysplastic gastric epithelial cells with enlarged, hyperchromatic and crowded nuclei were distributed in mucoderm. Differentiated size of epithelial cells and cavity fusion of gastric mucosa was exited. Thus, these results demonstrated that abnormality of gastric epithelial dysplasia had presented in gastric mucosa. Intestinal metaplasia and dysplasia had an improvement in positive group to a certain degree. Interestingly, the scope of dysplastic gastric epithelial cells was decreased after treatment with WPX, indicating WPX decoction could protect the gastric mucosa in MNNG-induced rats. Additionally, the degree of intestinal metaplasia was evaluated by HID-AB-PAS staining. Negative staining was confirmed in control group. By contrast, intestinal metaplasia cells were situated in gastric cavity side and lamina proprian in model group. However, positive group showed significant improvement. The areas of two subtypes of intestinal metaplasia were attenuated after treatment with WPX decoction, suggest that WPX decoction reversed MNNG-induced GPL.
peroxidase-conjugated (HRP) secondary antibody IgG at room temperature for 1.5 h. The immunoreactivity was detected by 5220 Multi (Tanon, China), and β‑actin was used to standardized the proteins expression. 2.6. Immunohistochemistry Streptavidin-peroxidase(SP) method was used to examine the expression of LDHA in gastric mucosa. Detailed process was described as a previous study [28]. Anti-LDHA(abcam) antibody was diluted at 1:200, and SP immunohistochemistry Kit(Lot, 1010149701) was purchased from MAXIM. Moreover, Images were captured at X200 magnification using an Olympus BX51 microscope. 2.7. Statistical analysis All the statistical analyses were analyzed by the Student's t-test or one-way ANOVA using SPSS 22.0 software. Values are reported as the means ± SME. Statistically significant difference was at P < 0.01 or P < 0.05 level. 3. Results
3.2. Effects of WPX on regulation miRNA and LDHA in GPL rats 3.1. Histological analysis To further evaluate the effect of WPX, the level of LDHA and miRNA was examined. Fig.2 shows the gene and protein expressions of LDHA were obviously increased in MNNG-induced rats, and level of miRNA34a was markedly decreased as compared with levels in control group (#P < 0.05, ##P < 0.01). After ten weeks treatment with both dosages of WPX decoction, results revealed that decreased gene and
Histological observation was a pivotal approach to assess the degree of gastric mucosal lesion. Fig.1A presented histological changes of the stomachs in 8th, 12th and 16th week in MNNG-induced rats. In 8th week, occurrence of diffuse vacuolar lesion indicated intestinal metaplasia of gastric mucosa, while intestinal metaplasia with scattered dysplastic
Fig. 1. Histopathological Changes of the Gastric Mucosa. (A) Process of histological changes after spontaneous intake of MNNG solution in 8th, 12th and 16th week were analyzed via HE staining (100x). (B) Histological analysis on the gastric tissues of different groups after the treatment of WPX decoction (100x). (C) Effects of WPX decoction on histological changes in MNNG-induced GPL rats (HID-AB-PAS staining) (100x). 3
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Fig. 2. Effects of WPX on regulation miRNA and LDHA in GPL rats. (A) The downregulation of miRNA-34a is more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). # P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (B) The upregulation of LDHA is more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). #P < 0.05 and ## P < 0.01versus the control group.*P < 0.05 and **P < 0.01 versus the model group. (C) Western Blot analysis of LDHA protein in the GPL rats after treatment of WPX. The upregulation of LDHA protein is more apparent in the gastric mucosa than control group. Quantification of the band intensities are presented in the adjacent graphs. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ## P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (D)Effect of WPX on the protein of LDHA in rats. The protein levels were conducted by IHC. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group.
revealed that the expressions of CD147, MCT4 and HIF-1α in the model group were significantly increased when compared with those of respective control group (#P < 0.05, ##P < 0.01). Notably, WPX decoction could down-regulate their levels as compared with levels in model group(*P < 0.05, **P < 0.01). Meanwhile, the genes expressions of CD147, MCT4 and HIF-1α in the model group were significantly increased and ten weeks treatment with both doses of WPX decoction markedly decreased their expressions(*P < 0.05, **P < 0.01) (Fig. 3).
protein expressions of LDHA and increased expression of miRNA-34a were observed compared with those of model group (*P < 0.05, **P < 0.01). 3.3. WPX regulated MCT4、CD147 and HIF-1α expression in GPL rats According to the previous findings, and in order to further evaluate the role of MCT4, CD147 and HIF-1α in GPL rats, gastric mucosal epithelial samples from GPL rats were subjected. Western blot analysis was used to quantify the protein levels of the above molecules. Results 4
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Fig. 3. WPX regulated MCT4、CD147 and HIF-1α expression in GPL rats. (A–C)The upregulation of MCT4、CD147 and HIF-1α are more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). #P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (D) Western Blot analysis of MCT4、CD147 and HIF-1α protein in the GPL rats after treatment of WPX. The upregulation of MCT4、CD147 and HIF-1α protein are more apparent in the gastric mucosa than control group. (E–H)Quantification of the band intensities are presented in the adjacent graphs. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ## P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group.
treatment with both doses of WPX decoction markedly decreased their expressions (*P < 0.05, **P < 0.01).
3.4. Effects of WPX on regulation glycolysis through PI3K/AKT pathway in GPL rats Based on the previous study, PI3K/Akt/mTOR signaling pathway is involved in the regulation of aerobic glycolysis which is crucial in tumor occurrence and development. Based on these findings, we decided to prove the role of PI3K/AKT pathway in GPL rats. Fluorescence quantitative PCR showed that compared with the model group, the expression of PI3K, Akt and mTOR in the group of WPX were significantly lower than the model group (*P < 0.05, **P < 0.01). Meanwhile, the protein levels of the above molecules were quantified by Western Blot analysis. Fig. 4 shows that the levels of PI3K, Akt and mTOR protein expressions of the model group significantly increased (#P < 0.05,##P < 0.01). The 10-week administration of WPX markedly decreased the expressions of PI3K, Akt and mTOR when compared with those of the model group (*P < 0.05, **P < 0.01). Fig. 5 shows the protein expressions of PI3K, AKT and mTOR were determined using immunohistochemistry. The values of the average optical density were used to evaluate protein levels in different groups. These molecules level significantly increased in model group when compared with control group(#P < 0.05,##P < 0.01). Ten weeks
4. Discussion The incidence of gastric carcinoma is a primary cause of mortality in the world, and the conventional therapies, including surgery, chemotherapy and radiation therapy, are not completely effective in curing gastric cancer [29]. By contrast, strategies of preventing gastric cancer is increasingly accepted and progressively recognized by patient, clinician and research. To date, curing GPL may be a both traditional treatment in China, which provides a novel sight in the prevention of occurrence and development of gastric cancer. According to Correa’s theory [30], gastric cancer is started with chronic active gastritis, followed by chronic atrophic gastritis, gastric intestinal metaplasia and gastric epithelial dysplasia. It has been fully evidenced that abnormal glycolysis is involved in the process of gastric cancer [5–7], and metabolic alterations including glycolysis are exited in Chronic atrophic gastritis [31]. Therefore, there is a great possibility that abnormal glycolysis exits in GPL. In the present study, we found that WPX can reverse MNNG-induced GPL by mediating glycolysis-relative molecules. 5
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Fig. 4. Effects of WPX on regulation glycolysis through PI3K/AKT pathway in GPL rats. (A–C) The upregulation of PI3K、AKT and mTOR are more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). #P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (D) Western Blot analysis of PI3K、AKT and mTOR protein in the GPL rats after treatment of WPX. The upregulation ofPI3K、AKT and mTOR protein are more apparent in the gastric mucosa than control group. (E–H) Quantification of the band intensities are presented in the adjacent graphs. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ## P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group.
energetics and growth of glycolytic tumors [33]. The increased expression of CD147 and MCT4 in tumors is in agreement with our observation that the GPL epithelium express more CD147 and MCT4. In our work, it was confirmed that WPX decoction could reverse GPL. The preliminary results of this study suggested that expression of CD147 and MCT4 is involved in abnormal glycolysis in GPL rats, and the GPL reversed mechanisms of WPX decoction may be associated with its inhibition in expression of CD147 and MCT4. HIF-1α is transcription factor which can regulate the expression of MCT4 [16]. It is important to highlight that HIF-1α was identified to overexpressed in preneoplastic and premalignant lesions in breast, colon, and prostate [44], which may give some clues that alterations of energy metabolism occurred in the transition from normal gastric mucosa to gastric cancer. The PI3K/AkT signaling pathway is pivotal in the regulation of energy metabolism in tumors. In human gastric carcinoma, PI3K/AkT signaling pathway is activated [45]. Consistant with tumors, the activation of PI3K/AkT pathway is involved in the procession of GPL [46]. mTOR is downstream of PI3K/AkT which can induce the expression of HIF-1α and LDHA to mediate glycolysis in tumors. Futhermore, in precancerous lesions of cervical lesions, mTOR signaling pathway is activated [47]. Interestingly, upregulation of miRNA-34a can induce gastric cancer cell death via modulating PI3K/AKT pathway [19]. These studies may implicate that miRNA-34a/PI3K/Akt/mTOR signaling pathway could play an important role in precancerous lesions. In the
miRNA34-a is a tumor suppressor, which is associated with various cancers [32]. In gastric cancer, miRNA-34a is found to suppress the growth, invasion and metastasis of cancer cells [33]. Consistent with the expression of miRNA-34a in cancers, its level was significantly attenuated in MNNG-induced GPL and was markedly increased in WPX group. LDHA is an rate-limiting enzyme which can catalyses the conversion of pyruvate to lactate in process of glycolysis [34,35]. Its upregulation was regarded as function of promoting gastric carcinoma development and progression [36]. In this study, WPX decoction could decreased the up-expression of LDHA induced by MNNG. Our results are in agreement with the abnormal glycolysis may have been exited in GPL. Furthermore, miRNA-34a could regulate glycolysis and tumor growth in breast and cervical cancer by directly targeting LDHA [20,21]. Thus WPX decoction may reverse GPL by regulating miRNA34a/ LDHA pathway. Fllowing the up-regulation of LDHA in cancers, overproduced lactate need to be exported by coexpression of MCT4 and CD147 considering intracellular accumulated lactate would lead to tumor cells death [37,38]. MCT4 is mainly expressed in glycolytic tissues [39], and studies showed MCT4 expression was significantly increased in a variety of cancers [40–42]. Importantly, a research demonstrated that MCT4 exhibited a high expression in intestinal metaplasia and dysplasia when compared with Normal epithelium [43]. To note that Co-expression of CD147 is needed for the activity MCT4, which is crucial for
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Fig. 5. Effects of WPX on regulation glycolysis through PI3K/AKT pathway in GPL rats. (A) Regulation of WPX decoction on PI3K, AKT and mTOR by immunocytochemistry assay. Images were captured by a fluorescence microscope, X200. (B) Image‑Pro Plus was used to calculate the values of the average optical density. Data are expressed as the means ± SEM, #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group.
Author contributions
current study, aberrant expression of PI3K, Akt and mTOR is observed in MNNG-induced GPL. The potential therapeutic mechanism of WPX decoction may be associated with its regulation in miRNA-34a/PI3K/ AkT /mTOR signaling pathway.
Author contributions Tiantian Cai designed the study and contributed to initial data analysis. Chengzhe Zhang, Xiaohui Zeng, Ziming Zhao and Yan Yan conducted the experiment. Xuhua Yu and Lei Wu provided helpful advice for the manuscript. Huafeng Pan and Lin Lin critically reviewed and revised the manuscript, and approved the final manuscript as submitted. All authors meet the ICMJE criteria for authorship.
5. Conclusion In conclusion, the present study indicates that WPX decoction reverse MNNG-induced GPL via regulating miRNA-34a/LDHA signaling pathway and miRNA-34a/PI3K/Akt/mTOR signaling pathway. These findings may have important implications for the prevention of gastric cancer.
Declaration of Competing Interest The authors declare that they have no competing interests. 7
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CRediT authorship contribution statement [17]
Tiantian Cai: Data curation, Formal analysis, Writing - original draft. Chengzhe Zhang: Supervision. Xiaohui Zeng: Supervision. Ziming Zhao: Supervision, Funding acquisition. Yan Yan: Supervision. Xuhua Yu: Writing - review & editing. Lei Wu: Writing - review & editing. Lin Lin: Writing - review & editing. Huafeng Pan: Funding acquisition, Project administration.
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[19]
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Acknowledgements
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This research was supported by the National Natural Science Foundation of China (No.81673946, 81473620, 81704043, 81804004), High level university construction project of China (No.A1AFD018171Z11034, No.A1-AFD018171Z11037), Natural Science Foundation of Guangdong Province China (2017A030313845). Project of Guangdong Traditional Chinese Medicine Bureau (20182004).
[22] [23]
[24]
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Protective effects of Weipixiao decoction against MNNG-induced gastric precancerous lesions in rats
T
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Tiantian Caia,d, Chengzhe Zhanga,b,c, Xiaohui Zengb,c, Ziming Zhaob,c, , Yan Yana, Xuhua Yud,e, ⁎⁎⁎ Lei Wud,e, Lin Lind,e, , Huafeng Pana,⁎ a
Guangzhou University of Chinese Medicine, Guangzhou, Guanghdong, 510000, China Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine, Guangzhou, Guanghdong, 510000, China c Guangdong Provincial Key Laboratory of Research and Development in Traditional Chinese Medicine, Guangzhou, Guanghdong, 510000, China d Department of Respiratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou,Guanghdong, 510000, China e Department of Respiratory Medicine, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou,Guanghdong, 510000, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Precancerous gastric lesions Glycolysis Weipixiao Rats
Gastric cancer is recognized as one of the most common cancer. In-depth research of gastric precancerous lesions (GPL) plays an important role in preventing the occurrence of gastric cancer. Meanwhile, traditional treatment provides a novel sight in the prevention of occurrence and development of gastric cancer. The current study was designed to assess the effects of therapy with Weipixiao (WPX) decoction on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced GPL rats and the underlying molecular mechanisms. After 10-weeks treatment, all rats were sacrificed. Histopathological changes of gastric tissue were assessed via hematoxylin-eosin (HE) and High-iron diamine-Alcian blue-Periodic acid-Schiff (HID-AB-PAS) staining. To be fully evidenced, RT-qPCR, Western blot and immunohistochemistry were used to detect the expressions of LDHA, CD147, HIF-1α, MCT4, PI3K, AKT, mTOR and miRNA-34a, which were crucial factors for evaluating GPL in the aspect of glycolysis pathogenesis. According to the results of HE and HID-AB-PAS staining, it could be confirmed that MNNG-induced GPL rats were obviously reversed by WPX decoction. Additionally, the increased gene levels of LDHA, CD147, MCT4, PI3K, AKT, mTOR and HIF-1α in model group were down-regulated by WPX decoction, while miRNA-34a expression was decreased and up-regulated by WPX decoction. The significantly increased protein levels of LDHA, CD147, MCT4, PI3K, AKT, mTOR and HIF-1α induced by MNNG were attenuated in rats treated with WPX decoction. In brief, the findings of this study imply that abnormal glycolysis in MNNG-induced GPL rats was relieved by WPX decoction via regulation of the expressions of LDHA, CD147, HIF-1α, MCT4, PI3K, AKT, mTOR and miRNA-34a.
1. Introduction Gastric cancer is recognized as one of the most common cancer [1]. China is one of the highest incidence regions of gastric cancer in the world [2] and the pathogenicity mechanism remains unclear. To date, surgical interventions, the primary choice for treatment of gastric carcinoma, are therapeutic in less than 40% of cases as gastric carcinoma patients are typically diagnosed at terminal stages [3]. Therefore, methods for early prevention and diagnosis of gastric cancer are required. The formation of gastric cancer contains consecutive steps, in which intestinal metaplasia and dysplasia are identified as gastric
precancerous lesions (GPL) [4]. In-depth research of GPL can provide a novel sight in preventing the occurrence of gastric carcinoma. Emerging researches have demonstrated that aerobic glycolysis which means cancer cells will produce a large amount of lactate regardless of the availability of oxygen is involved in gastric cancer [5–7]. There is a high possibility that abnormal glycolysis occurs in GPL and N-methylN'-nitro-N-nitrosoguanidine (MNNG) was chosen to induce GPL [8]. Thus, it is a novel point to further demonstrate whether abnormal glycolysis is involved in GPL. Glycolysis has been recognized as one of the hallmarks of cancers, thus we hypothesize that abnormal glycolysis occurs in GPL. Lactate
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Corresponding author at: 232 East Ring Road, Guangzhou University Town, Panyu District, Guangzhou, 510006, China. Corresponding author at: 111 Dade Road, Yuexiu District, Guangzhou, 510120, China. ⁎⁎⁎ Corresponding author at: Guangdong Province Engineering Technology Research Institute of Traditional Chinese Medicine, Guangzhou, Guanghdong 510000, China. E-mail addresses: [email protected] (L. Lin), [email protected] (H. Pan). ⁎⁎
https://doi.org/10.1016/j.biopha.2019.109427 Received 16 June 2019; Received in revised form 29 August 2019; Accepted 2 September 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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Table 1 The components of WeiPiXiao. Plant Latin name
Family
Local name
English name
Used part
A. membranaceus(Fisch.)Bge. Pseeudostellaria heterophylla(Miq.)Pax ex pax et Hoffm Atractylodes macrocephala Koidz. Salvia miltiorrhiza Oldenlandia
Leguminosae Caryophyllaceae compositae Labiatae Rubiaceae
Huang Qi Tai Zi Shen Bai Zhu Dan Shen Bai Hua She She Cao
Astragalus Radix Pseudostellariae Atractylodes macrocephala Salvia miltiorrhiza Bge. Oldenlandia diffusa(Willd.)Roxb.
Root Root Root Root Grass
previously [26,27]. 50 Rats intake of MNNG were used as model group and 10 rats were randomly divided into normal group which were administered routinely. At the 16th week the model group (n = 40), including model group, positive(WFC, 0.2 g/kg), high dose WPX group (SD, WPX 19.8 g/kg/d, n = 10) and low dose WPX group (SD, WPX 9.9 g/kg/d, n = 10). After treatment for 10 weeks, all rats were humanely euthanized with sodium pentobarbital (140 mg/kg i.p.) after 12 h fasting, and the stomachs were removed immediately.
dehydrogenase A (LDHA), an enzyme which catalyses the last step of glycolysis, has been presented to be overexpressed in various types of cancers and tumor cells [9–12]. Overexpressed LDHA was regarded to promote progression of gastric cancer [5]. Overproduction and intracellular accumulation of lactate which leads to tumor cells death can be exported by coexpression of monocarboxylate transporters 4(MCT4) and CD147 [13,14]. Additionally, the tendency of LDHA and MCT4 expression is mediated by hypoxia inducible factor-1α (HIF-1α) [15,16]. Therefore, it is possible that inhibiting LDHA expression may be a vital point in the prevention of GPL. PI3K-Akt-mTOR signaling pathway is involved in the regulation of aerobic glycolysis which is crucial in tumor occurrence and development. In this signaling pathway, the activation of PI3K can induce the expression of Akt which is the regulate mTOR level. Previous studies have indicated that mTOR can regulate HIF-1α to mediate cellular metabolism [17,18]. Also of note, miRNA-34, a tumour suppressor, is capable of mediating the expression of LDHA and PI3K/Akt signaling pathway [19–21]. Weipixiao(WPX) decoction is a traditional herbal formula as shown in Table 1, contained Astragalus, Radix Pseudostellariae, Atractylodes macrocephala, Salvia miltiorrhiza Bge, Oldenlandia diffusa(Willd.) Roxb. On the theory of traditional Chinese medicine, it is believed that WPX can reverse GPL via strengthening spleen, dispersing blood stasis and detoxicating. In a clinical study, it was established that WPX may ameliorate the symptoms of CAG patients, demonstrating that WPX had a clear therapeutic effect in protect the gastric mucosa [22,23]. In addition, our previous studies demonstrated that WPX protected the gastric mucosa of rats from exposure to MNNG by regulating Wnt Pathway-Associated Proteins [24]. However, the further mechanisms responsible for the curative effects of WPX on GPL remain to be explored. Therefore, in the present research, we aim to determine whether WPX reverse GPL via inhibiting glycolysis process.
2.3. Histological staining All the rats were sacrificed and the stomach tissue was collected and fixed in 10% neutral-buffered formalin over 12 h. Then the samples were dehydrated in alcohol and xylene respectively. After dehydration, the samples were embedded in paraffin. Serial sections (4–5 μm thick) were obtained from the stomach and stained with hematoxylin and eosin (H&E) and High-iron diamine-Alcian blue-Periodic acid-Schiff (HID-AB-PAS) staining using standard techniques to observe the histological changes of the gastric mucosa and the types of intestinal metaplasia. 2.4. Reverse transcription‑polymerase chain reaction (RT‑qPCR) Total RNA was isolated from the gastric tissue samples using TRIzol reagent (BEIJING DINGGUO CHANGSHENG BIOTECHNOLOGY CO, LTD). RNA(2 μg) was reverse transcribed into Complementary DNA (cDNA) using the iQ5 Multicolor Real-Time PCR Detection system(BIORAD, USA). Subsequently, Real-time PCR analysis was carried out via iQ5 Multicolor Real-Time PCR Detection system(BIO-RAD, USA). And the DNA bands were standardization to those of GAPDH. The primer pairs were as follows: GAPDH forward, 5'-TCTCTGCTCCTCCCTGTTCT3' and reverse, 5'-TACGGCCAAATCCGTTCACA-3'; LDH-A forward, 5'TTGTTGGGGTTGGTGC-3' and reverse, 5'-TCCCTCTTGCTGACGG-3'; MCT4 forward, 5'-CCAGGCCCACGGCAGGTTTC-3' and reverse, 5'-GCC ACCGTAGTCACTGGCCG-3'; CD147 forward, 5'-GGCACCATCGTAACC TCTGT-3' and reverse, 5'-TCTTTCCCACCTTGATCCTG-3'; HIF-1α forward, 5'-CCCCTACTATGTCGCTTTCTTG-3' and reverse, 5'-AGGTTTGT GCTGCCTTGTATG-3'; PI3K forward, 5'-ACTAACTGGACCCTTGGATGC CTAC-3' and reverse, 5'-CAGCCAAGAACTATTGGCAAC-TGA-3'; AKT forward, 5'-TAGGCATCCCTTCCTTACAGC-3' and reverse, 5'-CGCTCAC GAGACAGGTGGA-3'.
2. Materials and methods 2.1. Preparation and qualitative analysis of WPX WPX were provided by the First Affiliated Hospital of Guangzhou University of Chinese Medicine, it is composed of five Chinese herbal medicines. The components and dosage of WPX is described in the Table 1. The qualitative analysis of WPX had been reaffirmed by highperformance liquid chromatography (HPLC) [25]. Specific chromatograms of the standard substances and the WPX are also displayed in the supplemental materials. Positive drug Weifuchun(WFC, 0.2 g/kg) was purchased from Hangzhou hu qingyutang pharmaceutical co. LTD (Lot: 1606663).
2.5. Western Blot analysis Radioimmunoprecipitation assay (RIPA) lysis buffer(500 μl) contained protease inhibitors(5 μl) was used to extracted total protein from the frozen tissue(500 μg), and the protein concentration was determined by the bicinchoninic acid assay (BCA). Proteins were separated by SDS-PAGE and transferred into nitrocellulose membranes. Membranes were probed overnight with respective antibodies [β-actin (1:1000 dilution, sigma, CST124E); LDHA(1:1000 dilution, proteintech, 21799-1-AP); MCT4(1:1500 dilution, Santa cruz, sc-376140); CD1147(1:2000 dilution, abcam, ab108317); HIF-1α(1:1500 dilution, abcam, ab2185); PI3k(1:2000 dilution, abcam, ab151549); AKT(1:1000 dilution, abcam, ab8805); mTOR(1:2000 dilution, abcam, ab32028)]. Subsequently, goat anti-rabbit or goat anti-mouse horseradish
2.2. Materials and animals A total of 60 male Sprague-Dawley (SD) rats aged 4–6 weeks, 180–200 g of weight were obtained from Guangdong Medical Laboratory Animal Center(44007200027077). The entire study was performed with the permission and supervision of the Animal Ethics Committee of Guangdong Province Engineering Technology Research Institute of T.C.M.(GDPITCM160129). Mild GPL-like gastric disease was induced in SD rats by exposing to combination of MNNG solution for 200 mg/L and feeding every other day for 26 weeks as described 2
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cell occurred in 12th week. Meanwhile, the incidence of dysplasia was slightly higher in 16th week than 8th and 12th week. Fig.1B showed the histopathological changes of the gastric mucosa. By contrast to control group, it could be observed that the thickness of basement membrane were more asymmetrical in model group. In addition, dysplastic gastric epithelial cells with enlarged, hyperchromatic and crowded nuclei were distributed in mucoderm. Differentiated size of epithelial cells and cavity fusion of gastric mucosa was exited. Thus, these results demonstrated that abnormality of gastric epithelial dysplasia had presented in gastric mucosa. Intestinal metaplasia and dysplasia had an improvement in positive group to a certain degree. Interestingly, the scope of dysplastic gastric epithelial cells was decreased after treatment with WPX, indicating WPX decoction could protect the gastric mucosa in MNNG-induced rats. Additionally, the degree of intestinal metaplasia was evaluated by HID-AB-PAS staining. Negative staining was confirmed in control group. By contrast, intestinal metaplasia cells were situated in gastric cavity side and lamina proprian in model group. However, positive group showed significant improvement. The areas of two subtypes of intestinal metaplasia were attenuated after treatment with WPX decoction, suggest that WPX decoction reversed MNNG-induced GPL.
peroxidase-conjugated (HRP) secondary antibody IgG at room temperature for 1.5 h. The immunoreactivity was detected by 5220 Multi (Tanon, China), and β‑actin was used to standardized the proteins expression. 2.6. Immunohistochemistry Streptavidin-peroxidase(SP) method was used to examine the expression of LDHA in gastric mucosa. Detailed process was described as a previous study [28]. Anti-LDHA(abcam) antibody was diluted at 1:200, and SP immunohistochemistry Kit(Lot, 1010149701) was purchased from MAXIM. Moreover, Images were captured at X200 magnification using an Olympus BX51 microscope. 2.7. Statistical analysis All the statistical analyses were analyzed by the Student's t-test or one-way ANOVA using SPSS 22.0 software. Values are reported as the means ± SME. Statistically significant difference was at P < 0.01 or P < 0.05 level. 3. Results
3.2. Effects of WPX on regulation miRNA and LDHA in GPL rats 3.1. Histological analysis To further evaluate the effect of WPX, the level of LDHA and miRNA was examined. Fig.2 shows the gene and protein expressions of LDHA were obviously increased in MNNG-induced rats, and level of miRNA34a was markedly decreased as compared with levels in control group (#P < 0.05, ##P < 0.01). After ten weeks treatment with both dosages of WPX decoction, results revealed that decreased gene and
Histological observation was a pivotal approach to assess the degree of gastric mucosal lesion. Fig.1A presented histological changes of the stomachs in 8th, 12th and 16th week in MNNG-induced rats. In 8th week, occurrence of diffuse vacuolar lesion indicated intestinal metaplasia of gastric mucosa, while intestinal metaplasia with scattered dysplastic
Fig. 1. Histopathological Changes of the Gastric Mucosa. (A) Process of histological changes after spontaneous intake of MNNG solution in 8th, 12th and 16th week were analyzed via HE staining (100x). (B) Histological analysis on the gastric tissues of different groups after the treatment of WPX decoction (100x). (C) Effects of WPX decoction on histological changes in MNNG-induced GPL rats (HID-AB-PAS staining) (100x). 3
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Fig. 2. Effects of WPX on regulation miRNA and LDHA in GPL rats. (A) The downregulation of miRNA-34a is more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). # P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (B) The upregulation of LDHA is more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). #P < 0.05 and ## P < 0.01versus the control group.*P < 0.05 and **P < 0.01 versus the model group. (C) Western Blot analysis of LDHA protein in the GPL rats after treatment of WPX. The upregulation of LDHA protein is more apparent in the gastric mucosa than control group. Quantification of the band intensities are presented in the adjacent graphs. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ## P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (D)Effect of WPX on the protein of LDHA in rats. The protein levels were conducted by IHC. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group.
revealed that the expressions of CD147, MCT4 and HIF-1α in the model group were significantly increased when compared with those of respective control group (#P < 0.05, ##P < 0.01). Notably, WPX decoction could down-regulate their levels as compared with levels in model group(*P < 0.05, **P < 0.01). Meanwhile, the genes expressions of CD147, MCT4 and HIF-1α in the model group were significantly increased and ten weeks treatment with both doses of WPX decoction markedly decreased their expressions(*P < 0.05, **P < 0.01) (Fig. 3).
protein expressions of LDHA and increased expression of miRNA-34a were observed compared with those of model group (*P < 0.05, **P < 0.01). 3.3. WPX regulated MCT4、CD147 and HIF-1α expression in GPL rats According to the previous findings, and in order to further evaluate the role of MCT4, CD147 and HIF-1α in GPL rats, gastric mucosal epithelial samples from GPL rats were subjected. Western blot analysis was used to quantify the protein levels of the above molecules. Results 4
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Fig. 3. WPX regulated MCT4、CD147 and HIF-1α expression in GPL rats. (A–C)The upregulation of MCT4、CD147 and HIF-1α are more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). #P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (D) Western Blot analysis of MCT4、CD147 and HIF-1α protein in the GPL rats after treatment of WPX. The upregulation of MCT4、CD147 and HIF-1α protein are more apparent in the gastric mucosa than control group. (E–H)Quantification of the band intensities are presented in the adjacent graphs. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ## P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group.
treatment with both doses of WPX decoction markedly decreased their expressions (*P < 0.05, **P < 0.01).
3.4. Effects of WPX on regulation glycolysis through PI3K/AKT pathway in GPL rats Based on the previous study, PI3K/Akt/mTOR signaling pathway is involved in the regulation of aerobic glycolysis which is crucial in tumor occurrence and development. Based on these findings, we decided to prove the role of PI3K/AKT pathway in GPL rats. Fluorescence quantitative PCR showed that compared with the model group, the expression of PI3K, Akt and mTOR in the group of WPX were significantly lower than the model group (*P < 0.05, **P < 0.01). Meanwhile, the protein levels of the above molecules were quantified by Western Blot analysis. Fig. 4 shows that the levels of PI3K, Akt and mTOR protein expressions of the model group significantly increased (#P < 0.05,##P < 0.01). The 10-week administration of WPX markedly decreased the expressions of PI3K, Akt and mTOR when compared with those of the model group (*P < 0.05, **P < 0.01). Fig. 5 shows the protein expressions of PI3K, AKT and mTOR were determined using immunohistochemistry. The values of the average optical density were used to evaluate protein levels in different groups. These molecules level significantly increased in model group when compared with control group(#P < 0.05,##P < 0.01). Ten weeks
4. Discussion The incidence of gastric carcinoma is a primary cause of mortality in the world, and the conventional therapies, including surgery, chemotherapy and radiation therapy, are not completely effective in curing gastric cancer [29]. By contrast, strategies of preventing gastric cancer is increasingly accepted and progressively recognized by patient, clinician and research. To date, curing GPL may be a both traditional treatment in China, which provides a novel sight in the prevention of occurrence and development of gastric cancer. According to Correa’s theory [30], gastric cancer is started with chronic active gastritis, followed by chronic atrophic gastritis, gastric intestinal metaplasia and gastric epithelial dysplasia. It has been fully evidenced that abnormal glycolysis is involved in the process of gastric cancer [5–7], and metabolic alterations including glycolysis are exited in Chronic atrophic gastritis [31]. Therefore, there is a great possibility that abnormal glycolysis exits in GPL. In the present study, we found that WPX can reverse MNNG-induced GPL by mediating glycolysis-relative molecules. 5
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Fig. 4. Effects of WPX on regulation glycolysis through PI3K/AKT pathway in GPL rats. (A–C) The upregulation of PI3K、AKT and mTOR are more apparent in the gastric mucosa than control group. Data are shown as the mean ± SD (n = 3 rats per group). #P < 0.05 and ##P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group. (D) Western Blot analysis of PI3K、AKT and mTOR protein in the GPL rats after treatment of WPX. The upregulation ofPI3K、AKT and mTOR protein are more apparent in the gastric mucosa than control group. (E–H) Quantification of the band intensities are presented in the adjacent graphs. Data are shown as the mean ± SEM (n = 3 rats per group). #P < 0.05 and ## P < 0.01 versus the control group. *P < 0.05 and **P < 0.01 versus the model group.
energetics and growth of glycolytic tumors [33]. The increased expression of CD147 and MCT4 in tumors is in agreement with our observation that the GPL epithelium express more CD147 and MCT4. In our work, it was confirmed that WPX decoction could reverse GPL. The preliminary results of this study suggested that expression of CD147 and MCT4 is involved in abnormal glycolysis in GPL rats, and the GPL reversed mechanisms of WPX decoction may be associated with its inhibition in expression of CD147 and MCT4. HIF-1α is transcription factor which can regulate the expression of MCT4 [16]. It is important to highlight that HIF-1α was identified to overexpressed in preneoplastic and premalignant lesions in breast, colon, and prostate [44], which may give some clues that alterations of energy metabolism occurred in the transition from normal gastric mucosa to gastric cancer. The PI3K/AkT signaling pathway is pivotal in the regulation of energy metabolism in tumors. In human gastric carcinoma, PI3K/AkT signaling pathway is activated [45]. Consistant with tumors, the activation of PI3K/AkT pathway is involved in the procession of GPL [46]. mTOR is downstream of PI3K/AkT which can induce the expression of HIF-1α and LDHA to mediate glycolysis in tumors. Futhermore, in precancerous lesions of cervical lesions, mTOR signaling pathway is activated [47]. Interestingly, upregulation of miRNA-34a can induce gastric cancer cell death via modulating PI3K/AKT pathway [19]. These studies may implicate that miRNA-34a/PI3K/Akt/mTOR signaling pathway could play an important role in precancerous lesions. In the
miRNA34-a is a tumor suppressor, which is associated with various cancers [32]. In gastric cancer, miRNA-34a is found to suppress the growth, invasion and metastasis of cancer cells [33]. Consistent with the expression of miRNA-34a in cancers, its level was significantly attenuated in MNNG-induced GPL and was markedly increased in WPX group. LDHA is an rate-limiting enzyme which can catalyses the conversion of pyruvate to lactate in process of glycolysis [34,35]. Its upregulation was regarded as function of promoting gastric carcinoma development and progression [36]. In this study, WPX decoction could decreased the up-expression of LDHA induced by MNNG. Our results are in agreement with the abnormal glycolysis may have been exited in GPL. Furthermore, miRNA-34a could regulate glycolysis and tumor growth in breast and cervical cancer by directly targeting LDHA [20,21]. Thus WPX decoction may reverse GPL by regulating miRNA34a/ LDHA pathway. Fllowing the up-regulation of LDHA in cancers, overproduced lactate need to be exported by coexpression of MCT4 and CD147 considering intracellular accumulated lactate would lead to tumor cells death [37,38]. MCT4 is mainly expressed in glycolytic tissues [39], and studies showed MCT4 expression was significantly increased in a variety of cancers [40–42]. Importantly, a research demonstrated that MCT4 exhibited a high expression in intestinal metaplasia and dysplasia when compared with Normal epithelium [43]. To note that Co-expression of CD147 is needed for the activity MCT4, which is crucial for
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Fig. 5. Effects of WPX on regulation glycolysis through PI3K/AKT pathway in GPL rats. (A) Regulation of WPX decoction on PI3K, AKT and mTOR by immunocytochemistry assay. Images were captured by a fluorescence microscope, X200. (B) Image‑Pro Plus was used to calculate the values of the average optical density. Data are expressed as the means ± SEM, #P < 0.05 and ##P < 0.01 compared with the control group; *P < 0.05 and **P < 0.01 compared with the model group.
Author contributions
current study, aberrant expression of PI3K, Akt and mTOR is observed in MNNG-induced GPL. The potential therapeutic mechanism of WPX decoction may be associated with its regulation in miRNA-34a/PI3K/ AkT /mTOR signaling pathway.
Author contributions Tiantian Cai designed the study and contributed to initial data analysis. Chengzhe Zhang, Xiaohui Zeng, Ziming Zhao and Yan Yan conducted the experiment. Xuhua Yu and Lei Wu provided helpful advice for the manuscript. Huafeng Pan and Lin Lin critically reviewed and revised the manuscript, and approved the final manuscript as submitted. All authors meet the ICMJE criteria for authorship.
5. Conclusion In conclusion, the present study indicates that WPX decoction reverse MNNG-induced GPL via regulating miRNA-34a/LDHA signaling pathway and miRNA-34a/PI3K/Akt/mTOR signaling pathway. These findings may have important implications for the prevention of gastric cancer.
Declaration of Competing Interest The authors declare that they have no competing interests. 7
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CRediT authorship contribution statement [17]
Tiantian Cai: Data curation, Formal analysis, Writing - original draft. Chengzhe Zhang: Supervision. Xiaohui Zeng: Supervision. Ziming Zhao: Supervision, Funding acquisition. Yan Yan: Supervision. Xuhua Yu: Writing - review & editing. Lei Wu: Writing - review & editing. Lin Lin: Writing - review & editing. Huafeng Pan: Funding acquisition, Project administration.
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[19]
[20]
Acknowledgements
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This research was supported by the National Natural Science Foundation of China (No.81673946, 81473620, 81704043, 81804004), High level university construction project of China (No.A1AFD018171Z11034, No.A1-AFD018171Z11037), Natural Science Foundation of Guangdong Province China (2017A030313845). Project of Guangdong Traditional Chinese Medicine Bureau (20182004).
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