Driving factors analysis of spatial-temporal evolution of vegetation ecosystem in rocky desertification restoration area of Guizhou Province, China.

The investigation of the temporal-spatial characteristics and driving factors of vegetation ecosystem (VE) alterations held significant practical implications for the evaluation of the efficacy of rocky desertification management initiatives and safeguarding the ecological environment in the rocky desertification restoration region of Guizhou. We computed the comprehensive ecological quality index (Q) of vegetation based on the normalized difference vegetation index (NDVI) and net primary productivity (NPP). Combined with temperature, precipitation, sunshine duration, rocky desertification grade, land use, and the time series of various regions being included in national ecological functional zones, we analyzed the spatial-temporal distribution characteristics of VE changes and their response to climate change (CC) and ecological engineering (EE) by using partial derivative analysis method and scenario setting method in rocky desertification restoration areas in Guizhou. Results demonstrated that (1) the average values of NDVI, NPP, and Q all showed a fluctuating upward trend since 2000. Although the VE status of rocky desertification area was obviously worse than that of no rocky desertification area, it has a higher growth rate, especially the growth rates of NDVI, NPP, and Q in severe rocky desertification area were as high as 0.0050 year-1, 9.0733 g C m-2 year-1, and 0.7829 year-1, and the area with high recovery degree accounted for 93.19%, followed by the middle rocky desertification area. (2) CC was the main driving factor for NDVI and Q recovery, and EE was the main driving factor for NPP recovery. The contribution of EE to NPP and Q recovery increased with the increase of rocky desertification, as high as 82.13% and 30.31% in severe rocky desertification area. (3) The more serious the rocky desertification was, the more dependent the vegetation restoration was on ecological engineering, and the more difficult the restoration was. It was urgent to solve the ecological environmental problems. (4) EE played a greater role in the restoration of VE in the early stage of implementation. Its role gradually decreased in the later stages of implementation, while the role of CC increased. We provide a scientific basis for the follow-up treatment of rocky desertification, ecological environment restoration, and ecological protection effectiveness evaluation in Guizhou.

Morchella esculenta cultivation in fallow paddy fields and drylands affects the diversity of soil bacteria and soil chemical properties.

The properties of paddy field (DT) and dry land (HD) soil and food production can be enhanced by the cultivation of Morchella esculenta (ME) during the fallow period. However, whether ME cultivation affects the soil health and microbial diversity of paddy fields and drylands during the cultivation period remains unclear, and this has greatly limited the wider use of this cultivation model. Here, we analyzed the soil chemical properties and bacterial diversity (via metabarcoding sequencing) of DT and HD soils following ME cultivation. Our findings indicated that ME cultivation could enhance soil health. The content of soil phosphorus and potassium (K) was increased in DT soil under ME cultivation, and the K content was significantly higher in HD soil than in DT soil under ME cultivation. ME cultivation had a weak effect on alpha diversity, and ME cultivation affected the abundance of some genera of soil bacteria. The cultivation of ME might reduce the methane production capacity of DT soil and enhance the nitrogen cycling process of HD soil based on the results of functional annotation analysis. Network analysis and correlation analysis showed that Gemmatimonas, Bryobacter, and Anaeromyxobacter were the key bacterial genera regulating soil chemical properties in DT soil under ME cultivation, and Bryobacter, Bacillus, Streptomyces, and Paenarthrobacter were the key taxa associated with the accumulation of K in HD soil. The results of our study will aid future efforts to further improve this cultivation model.

Quercetin protects against LPS-induced lung injury in mice via SIRT1-mediated suppression of PKM2 nuclear accumulation.

The role of NOD-like receptor protein 3 (NLRP3)-mediated macrophages pyroptosis in acute lung injury (ALI) is well-established. Quercetin (Que) is a natural bioflavonoid compound with anti-inflammatory and antioxidative properties that reportedly inhibits the NLRP3 inflammasome in sepsis-induced organ dysfunctions such as ALI. However, the mechanism underlying the inhibitory effect of quercetin on NLRP3 activation remains unclear. In this study, we established an endotoxin-induced ALI mouse model with an in vitro LPS challenge. We demonstrated that the administration of quercetin could significantly reduce pulmonary injury and decrease the production of pro-inflammatory cytokines. Moreover, we found that quercetin could inhibit the activation of the NLRP3 inflammasome by suppressing the nuclear accumulation of PKM2 and increasing SIRT1 levels. Importantly, treatment with SRT1720 (a specific SIRT1 activator) could inhibit the nuclear accumulation of PKM2 and the activation of NLRP3. Besides, preventing PKM2 dimerization with ML265 yielded an anti-inflammatory effect, similar to findings observed for SRT1720. In addition, we found that SIRT1 silencing or inhibition by EX527 could increase NLRP3 activation and nuclear accumulation of PKM2 and override quercetin-mediated anti-inflammatory activity. These findings indicated that quercetin could downregulate NLRP3 inflammasome activation by inhibiting the nuclear accumulation of PKM2 and upregulating SIRT1 expression, expanding the treatment landscape for ARDS.

Detecting Key Functional Components Group and Speculating the Potential Mechanism of Xiao-Xu-Ming Decoction in Treating Stroke.

Stroke is a cerebrovascular event with cerebral blood flow interruption which is caused by occlusion or bursting of cerebral vessels. At present, the main methods in treating stroke are surgical treatment, statins, and recombinant tissue-type plasminogen activator (rt-PA). Relatively, traditional Chinese medicine (TCM) has widely been used at clinical level in China and some countries in Asia. Xiao-Xu-Ming decoction (XXMD) is a classical and widely used prescription in treating stroke in China. However, the material basis of effect and the action principle of XXMD are still not clear. To solve this issue, we designed a new system pharmacology strategy that combined targets of XXMD and the pathogenetic genes of stroke to construct a functional response space (FRS). The effective proteins from this space were determined by using a novel node importance calculation method, and then the key functional components group (KFCG) that could mediate the effective proteins was selected based on the dynamic programming strategy. The results showed that enriched pathways of effective proteins selected from FRS could cover 99.10% of enriched pathways of reference targets, which were defined by overlapping of component targets and pathogenetic genes. Targets of optimized KFCG with 56 components can be enriched into 166 pathways that covered 80.43% of 138 pathways of 1,012 pathogenetic genes. A component potential effect score (PES) calculation model was constructed to calculate the comprehensive effective score of components in the components-targets-pathways (C-T-P) network of KFCGs, and showed that ferulic acid, zingerone, and vanillic acid had the highest PESs. Prediction and docking simulations show that these components can affect stroke synergistically through genes such as MEK, NFκB, and PI3K in PI3K-Akt, cAMP, and MAPK cascade signals. Finally, ferulic acid, zingerone, and vanillic acid were tested to be protective for PC12 cells and HT22 cells in increasing cell viabilities after oxygen and glucose deprivation (OGD). Our proposed strategy could improve the accuracy on decoding KFCGs of XXMD and provide a methodologic reference for the optimization, mechanism analysis, and secondary development of the formula in TCM.

The NF-κB/miR-488/ERBB2 axis modulates pancreatic cancer cell malignancy and tumor growth through cell cycle signaling.

Pancreatic cancer is one of the malignancies having the poorest prognosis due to late diagnoses and lack of efficient treatment regimens. The identification of potential miRNA-targeted gene axes could act as targets for developing novel treatment strategies. Herein, it was assessed that miR-488 expression was markedly downregulated within pancreatic carcinoma. Higher expression of miR-488 was shown to be linked to better prognosis rates of pancreatic carcinoma as per online data. Within two pancreatic tumor cells, MIA PaCa-2 and PANC-1, miR-488 overexpression significantly suppressed malignant cytological behavior by inhibiting cell viability, enhancing cell apoptosis, and inducing cell cycle G2/M-phase arrest. Moreover, miR-488 overexpression also decreased the protein levels of cell cycle regulators, including cyclin A, cyclin B, CDK1, and CDK2. miR-488 directly targets ERBB2 (receptor tyrosine-protein kinase2) to suppress the expression of ERBB2 by targeting its 3'UTR. ERBB2 knockdown in MIA PaCa-2 and PANC-1 cell lines suppressed, but miR-488 inhibition enhanced the cancer cell biological malignant behavior; the effects of miR-488 inhibition on pancreatic cancer cells were significantly reversed by ERBB2 knockdown. NF-κB suppressed the expression of miR-488 transcriptionally via targeting its promoter region, consequentially repressing the tumor-suppressive effects of miR-488 upon pancreatic tumor cells. Thus, an NF-κB/miR-488/ERBB2 axis modulating pancreatic cancer cell malignancy and tumor growth through cell cycle signaling was conclusively demonstrated.

DSCR9/miR-21-5p axis inhibits pancreatic cancer proliferation and resistance to gemcitabine via BTG2 signaling.

The outcome of pancreatic adenocarcinoma (PAAD) patients is poor, given resistance to gemcitabine. Long noncoding RNA (lncRNA) has been implicated in the carcinogenesis of pancreatic cancer; however, its function and mechanism in PAAD resistance to gemcitabine (GEM) are yet unknown. Herein, we demonstrate that lncRNA DSCR9 is significantly reduced in PAAD in vitro and in vivo. CCK-8, BrdU and flow cytometry assays show that overexpression of DSCR9 markedly suppresses pancreatic cancer cell proliferation and invasion, and promotes apoptosis under gemcitabine treatment. BTG2 acts as a tumor suppressor by reducing the proliferation and invasion of pancreatic cancer cells and increasing gemcitabine-induced apoptosis. Immunofluorescence (IF) staining combined with fluorescence in situ hybridization (FISH) of pancreatic cancer tissues shows that DSCR9 and BTG2 are both increased in pancreatic cancer tissues. Luciferase assay shows that miR-21-5p simultaneously binds to DSCR9 and 3'UTR of BTG2; DSCR9 relieves miR-21-5p-induced inhibition of BTG2 by competing with BTG2 for miR-21-5p binding. Overexpression of miR-21-5p enhances the invasiveness of pancreatic cancer cells by promoting cancer cell proliferation and invasion and attenuating gemcitabine-induced apoptosis. Overexpression of miR-21-5p attenuates the effect of DSCR9 overexpression on BTG2 expression and invasiveness of pancreatic cancer cells. Finally, miR-21-5p expression is increased, while BTG2 expression is decreased in pancreatic cancer tissues. miR-21-5p is negatively correlated with DSCR9 and BTG2. In conclusion, the DSCR9/miR-21-5p/BTG2 axis modulates pancreatic cancer proliferation, invasion, and gemcitabine resistance.

Role of HMGB1 in TNF-α Combined with Z-VAD-fmk-Induced L929 Cells Necroptosis.

The present study established a necroptosis model in vitro and investigated the role of HMGB1 in cell necroptosis. A combination of tumor necrosis factor-α and z-VAD-fmk was used to induce necroptosis in L929 cells with necroptosis inhibitor necrostatin-1 applied as an intervention. Flow cytometry and transmission electron microscopy (TEM) were used to measure cell necroptosis. Western blotting assay was applied to detect the expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL) and HMGB1. Co-immunoprecipitation (Co-IP) assay was used to confirm the interaction between HMGB1 and RIPK3. Our study demonstrated that HMGB1 migrated from the nucleus to the cytoplasm at the onset of necroptosis and was subsequently released passively to the extracellular matrix. Further experiments determined that the binding of HMGB1 with RIPK3 in the cytoplasm was loose during necroptosis. By contrast, when necroptosis was inhibited, the interaction in the cytoplasm was tight suggesting that this association between HMGB1 and RIPK3 might affect its occurrence. In conclusion, the transfer of HMGB1 from nucleus to cytoplasm, and its interaction with RIPK3 might be potentially involved in necroptosis.

Corrigendum: Detecting key functional components group and speculating the potential mechanism of Xiao-Xu-Ming decoction in treating stroke.

[This corrects the article DOI: 10.3389/fcell.2022.753425.].

Effects of enzymatic hydrolysis on physicochemical property and antioxidant activity of mulberry (Morus atropurpurea Roxb.) leaf protein.

To improve the antioxidant efficiency of mulberry leaf protein (MLP), alcalase, protamex, papain, flavourzyme, neutrase, and trypsin were used to hydrolyze MLP. The yield of soluble peptides, secondary structures, molecular weight distributions, and antioxidant activities of MLP hydrolysates (MLPHs) were investigated. Results showed that the native MLP was rich in the fraction above 6.5 kDa and was mainly composed of ß-sheets, while MLPHs were abundant in the fractions of 0.3-0.6 kDa and 0.6-6.5 kDa and were mainly composed of disordered coils and ß-folds. Limited hydrolysis of MLP could lead to better antioxidant activity than extensive hydrolysis. After enzymatic hydrolysis, the content of total sugar and total phenol in MLP increased significantly. MLP hydrolysates prepared with neutrase, alcalase, and protamex were preferable to other enzymes. Meanwhile, an enzyme to substrate level of 1% and a hydrolysis time of 2 hr were the optimum conditions to obtain higher antioxidant hydrolysates using neutrase.

Nomogram Incorporating the WNT/ß-Catenin Signaling Pathway for Predicting the Survival of Cutaneous Melanoma.

BACKGROUND: Accurate prediction of the survival of cutaneous melanoma (CM) permits the selection of the optimal treatment. Currently, the TNM stage has limitations in predicting the survival of CM. There is evidence that the WNT/ß-catenin signaling pathway has the potential to predict the CM prognosis. However, it still needs further investigation. OBJECTIVE: This study aims to establish a nomogram incorporating the WNT/ß-catenin signaling pathway to improve the predicted accuracy of the overall survival (OS) of CM. METHODS: Two hundred and eighty CM patients were recruited and followed up. The clinicopathological characteristics and the key genes of the WNT/ß-catenin signaling pathway (VEGF, ß-catenin, and DKK1) were chosen as potential variables associated with the OS. In the training cohort (n = 190), a nomogram was built to estimate the 1-, 3-, and 5-year OS, and its discriminations and calibrations were valid by the verification cohort (n = 90). The predicted accuracies of the nomogram with or without the Wnt/ß-catenin pathway and TNM stage were compared. RESULTS: A nomogram integrating independent risk factors (ulceration, lymph node metastasis, distant metastasis, Breslow thickness, dermal mitoses, ß-catenin, VEGF, and DKK1), which were evaluated by a multivariate analysis, was constructed to predict the 1-, 3-, and 5-year OS of CM patients. Good discrimination and calibration were obtained regardless of the training or validation datasets. The nomogram incorporating the Wnt/ß-catenin signaling pathway showed the highest accuracy [area under the curve (AUC)=0.914, 0.852, 0.785] compared with the nomogram without the Wnt/ß-catenin signaling pathway (AUC=0.693, 0.640, 0.615) and the TNM stage (AUC=0.726, 0.693, 0.673). CONCLUSION: The prognostic value of the established nomogram incorporating the WNT/ß-catenin signaling pathway was better than it without WNT/ß-catenin signaling pathway and TNM stage, which might be beneficial in the development of optimal treatment options.

The Effects of Bioactive Compounds from Blueberry and Blackcurrant Powder on Oat Bran Pastes: Enhancing In Vitro Antioxidant Activity and Reducing Reactive Oxygen Species in Lipopolysaccharide-Stimulated Raw264.7 Macrophages.

In this study, blueberry and blackcurrant powder were chosen as the phenolic-rich enrichments for oat bran. A Rapid Visco Analyser was used to form blueberry and blackcurrant enriched oat pastes. An in vitro digestion process evaluated the changes of phenolic compounds and the in vitro antioxidant potential of extracts of pastes. The anthocyanidin profiles in the extracts were characterised by the pH differential method. The results showed that blueberry and blackcurrant powder significantly increased the content of phenolic compounds and the in vitro antioxidant capacity of pastes, while the total flavonoid content decreased after digestion compared to the undigested samples. Strong correlations between these bioactive compounds and antioxidant values were observed. Lipopolysaccharide-stimulated RAW264.7 macrophages were used to investigate the intracellular antioxidant activity of the extracts from the digested oat bran paste with 25% enrichment of blueberry or blackcurrant powder. The results indicated that the extracts of digested pastes prevented the macrophages from experiencing lipopolysaccharide (LPS)-stimulated intracellular reactive oxygen species accumulation, mainly by the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway. These findings suggest that the bioactive ingredients from blueberry and blackcurrant powder enhanced the in vitro and intracellular antioxidant capacity of oat bran pastes, and these enriched pastes have the potential to be utilised in the development of the functional foods.

Long non-coding RNA SPATA31D5P promoting proliferation, migration and invasion of breast cancer cells through adsorption of microRNA-320a / 解剖学报

Objective Long non-coding RNA(lncRNA) are aberrantly expressed in breast cancer(BC) and strongly associated with its survival prognosis. The aim of this study is to investigate the expression and effect of IncRNA SPATA31D5P on the invasion and migration capacity of breast cancer cells through adsorption of miR-320a. Methods Totally 30 cases of BC tissues and paraneoplastic tissues were collected, and the expression levels of SPATA31D5P in BC tissues and BC cell lines were detected by Real-time PCR. MDA-MB-231 cells were transfected with SPATA31D5P siRNA interference vector, and cell proliferation, invasion and migration capacity were determined using the cell counting kit-8 assay (CCK-8), 5-ethynyl-2'- deoxyuridine(EdU), Transwell and wound-healing assay respectively. And cell cycle and apoptosis were detected by flow cytometry. Bioinformatics approachs were used to screen for miRNAs that could bind complementarily to SPATA31D5P, and the regulatory effect of SPATA31D5P on miR-320a was detected by Real-time PCR and dual luciferase reporter assay. Results SPATA31D5P levels were significantly higher in BC tissues than in adjacent normal breast tissues, and SPATA31D5P expression was higher in each BC cell line than in normal breast epithelial cells MCF10 A. The level of SPATA31D5P in the interference group was 0. 288±0. 052, which was lower than that of the blank control group 1. 114±0. 096 and negative control (NC) group 1. 079±0. 128 (P< 0. 01). The proliferation activity of MDA- MB-231 cells in the interfered group was significantly reduced and apoptotic rate was obviously increased compared to the NC and control groups (P<0. 01) ;the Gj phase block was observed in the interfered group; the scratch healing rate and number of perforated cells in the interference group were (14. 36 ± 1. 75) % and (26±1.52), which were lower than (52. 25± 1.87)% and ( 67. 33 ± 2. 91 ) of the NC group (PcO.Ol). Dual luciferase experiments confirmed that SPATA31D5P could directly regulate miR-320a expression and luciferase activity. Conclusion SPATA31D5P is highly expressed in BC, interfering with SPATA31D5P expression effectively inhibits the proliferation, migration and invasion of MDA-MB-231 cells, and the mechanism may be related to the targeted regulation of miR-320a.

The effects of bioactive compounds from blueberry and blackcurrant powders on the inhibitory activities of oat bran pastes against α-amylase and α-glucosidase linked to type 2 diabetes.

The α-amylase and α-glucosidase inhibitory activities by extracts of oat bran, blueberry and blackcurrant powders, as well as oat bran pastes supplemented 25% of blueberry and blackcurrant powder, were studied by measuring their half inhibitory (IC50) concentrations. Addition of blueberry or blackcurrant powder into oat bran paste increased α-amylase and α-glucosidase inhibitory activities with a decrease in IC50 values. The main anthocyanidin content was measured by pH differential method and the potential inhibitory mechanisms of these extracts were also investigated by detailed inhibition kinetics and docking simulations. The results showed that: (1) cyanidin and delphinidin were the main anthocyanidin profiles in extracts; (2) only blackcurrant powder was a competitive inhibitor, while other extracts were all mixed-type inhibitors against α-amylase; (3) both blueberry- and blackcurrant-enriched pastes were competitive inhibitors, while other extracts were all mixed-type inhibitors towards α-glucosidase; (4) the α-amylase and α-glucosidase inhibitory activities by extracts were potentially driven by hydrogen bonding, cyanidin-3-glucoside and delphinidin-3-glucoside had stronger binding affinity compared to malvidin-3-glucoside and cyanidin-3-rutinside. This study suggested supplementary of blueberry and blackcurrant with oat bran might be a potential source of bioactive products for antidiabetic activity.

Anti-Inflammatory and Intestinal Microbiota Modulation Properties of Jinxiang Garlic (Allium sativum L.) Polysaccharides toward Dextran Sodium Sulfate-Induced Colitis.

Garlic polysaccharides are great potential agents because of their anti-inflammation, antioxidation, and immunomodulation properties. However, few studies have reported their anti-inflammatory effects on improving the colon system and corresponding intestinal microbiota. Herein, a water-soluble garlic polysaccharide (WSGP) was extracted from Jinxiang garlic to evaluate its effects on ameliorating dextran sulfate sodium (DSS)-induced colitis in a mouse model. The results showed that (1) after administration of the WSGP (200 or 400 mg/kg/day), the feed intake, body weight, and colon length of colitic mice were increased, while the disease activity index and the histological score of colitic mice were decreased; (2) the WSGP reduced the colonic tissue damage and inhibited the expression of inflammatory factors (interleukin 6, interleukin 1 beta , and tumor necrosis factor alpha); and (3) the WSGP enhanced the production of short-chain fatty acids and improved the composition of intestinal microbiota. The key microorganisms, including Muribaculaceae, Lachnospiraceae, Lachnospiraceae_NK4A136_group, Mucispirillum, Helicobacter, Ruminococcus_1, and Ruminiclostridium_5, were identified to be associated with inflammatory bowel diseases. Taken together, this study proved that WSGP supplementation could alleviate DSS-induced colitis by improving mucosal barriers, blocking proinflammatory cytokines, and modulating gut microbiota.

Expression of polypeptide N-acetylgalactosaminyltransferase-10 in lung cancer / 解剖学报

Objective The purpose of this study is to investigate the expression of the polypeptide N-acetylgalactosaminyltransferase-10 (ppGalNAc-T10) protein in lung and breast and colon cancer. Methods Immunohistochemistry was used to analyse ppGalNAc-T10 expressions in 110 lung cancer tissues, 100 breast cancer tissues and 110 colon cancer tissues. Meanwhile, different cancer has 10 cases of normal tissues as control. Results The expression of ppGalNAc-T10 in normal lung tissue was higher compared with that in lung cancer organizations, and ppGalNAc-T10 positive expression was found to be significantly associated with invasion depth of tumor (P < 0. 01). PpGalNAc-T10 protein expression in breast and colon cancer was no significantly difference than that in non-tumor breast and colon tissue. Conclusion PpGalNAc-T10 expression is a useful marker in lung cancer.

Correction: MiR-4524b-5p/WTX/ß-catenin axis functions as a regulator of metastasis in cervical cancer.

[This corrects the article DOI: 10.1371/journal.pone.0214822.].

MiR-4524b-5p/WTX/ß-catenin axis functions as a regulator of metastasis in cervical cancer.

Cervical cancer is the second most deadly gynecological tumor worldwide. MicroRNAs (miRNAs) play very important roles in tumor oncogenesis and progression. The mechanism of post-transcription regulation of WTX gene is still unknown. A series of differential miRNAs were discovered by microarray analysis comparing three pairs of primary cervical cancer specimens and their relapsed tumors from three patients. Quantitative reverse transcriptase PCR (qRT-PCR), Western Blot (WB) and Immunohistochemistry (IHC) was used to detect the expression of miR-4524b-5p and WTX in cervical cell lines and tissues. The biological function of miR-4524b-5p and WTX was investigated through knockdown and overexpression with inhibitor/siRNA and mimic/plasmid in vitro and in vivo. In this study, we found that miR-4524b-5p is highly expressed in relapsed cervical cancer specimens. Combined in vitro and in vivo experiments, showed that miR-4524b-5p could regulate the migration and invasion ability of cervical cancer. Furthermore, we also found that miR-4524b-5p could regulate the migration and invasion of cervical cancer by targeting WTX and that WTX could regulate the expression of ß-catenin. Taken together, our data identified a miR-4524b-5p/WTX/ß-catenin regulatory axis for cervical cancer, and miR-4524b-5p may be a potential target for cervical cancer therapy.

Exendin-4 impairs the autophagic flux to induce apoptosis in pancreatic acinar AR42J cells by down-regulating LAMP-2.

This study aimed to explore the mechanism of impaired autophagy flux induced by exendin-4 and its role on cell apoptosis in pancreatic AR42J cells. The AR42J cells were treated with various concentration of exendin-4 for several time points to assess its cytotoxicity by MTT assay. Then the AR42J cells were treated by 10pM exendin-4 for 72 h, the cell death was analyzed by flow cytometry and caspase-3 level was examined by Western blot with or without the pretreatment of z-VAD-fmk to testify whether exendin-4 induces the cell apoptosis. The protein levels of LC3B, p62 and LAMP-2 were assessed by Western blot, the mRNA level of LAMP-2 was quantified by quantitative PCR in the absence or presence of LAMP-2 over-expression plasmid and the expression and activity of CatB and CatL were tested by ELISA or activity assay methods in AR42J cells treated by exendin-4. The normal rats and the diabetes-model rats by high-fat and high-sugar diet for two month then with streptozotocin intraperitoneally were subcutaneously injected with exendin-4 for 10 weeks to test the expression of LAMP-2 mRNA and protein in the pancreas. Cells pretreated with Bafilomycin A1 were detected for LC3B and p62 expressions by Western blot. Cells pretreated by 3-MA were used to assess whether 3-MA can protect from exendin-4 cytotoxicity. We found that exendin-4 can decrease the AR42J cell viability as well as increase the cell death and cleaved caspase-3 level, which all can be inhibited by z-VAD-fmk. Exendin-4 can downregulate the expression of LAMP-2 and then impair the autophagy flux to induce the accumulation of LC3B-II and p62, but cannot change the expression and activity of CatB and CatL. Bafilomycin A1 almostly have no impact on the change of LC3B and p62 protein levels induced by exendin-4. Both 3-MA and overexpressed LAMP-2 can reduce the cytotoxicity of exendin-4. Therefore, we considered the down-regulation of LAMP-2 which can impair the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes to induce the AR42J cell apoptosis as the potential mechanism of chronic pancreatitis induced by exendin-4.

Exenatide Induces Impairment of Autophagy Flux to Damage Rat Pancreas.

OBJECTIVES: The study aimed to explore the alteration of autophagy in rat pancreas treated with exenatide. METHODS: Normal Sprague-Dawley rats and diabetes-model rats induced by 2-month high-sugar and high-fat diet and streptozotocin injection were subcutaneously injected with exenatide, respectively, for 10 weeks, with homologous rats treated with saline as control. Meanwhile, AR42J cells, pancreatic acinar cell line, were cultured with exenatide at doses of 5 pM for 3 days. The pancreas was disposed, and several sections were stained with hematoxylin-eosin. Immunohistochemistry was used to measure the expressions of glucagon-like peptide 1 receptor (GLP-1R) and cysteine-aspartic acid protease-3 in rat pancreas, and Western blot was used to test the expressions of GLP-1R, light chain 3B-I and -II, and p62 in rat pancreas and AR42J cells. The data were expressed as mean (standard deviation) and analyzed by unpaired Student's t-test. RESULTS: Exenatide can induce pathological changes in rat pancreas. The GLP-1R, p62, light chain 3B-II, and cysteine-aspartic acid protease-3 in rat pancreas and AR42J cells treated with exenatide were significantly overexpressed. CONCLUSIONS: Exenatide can activate and upregulate its receptor, GLP-1R, then impair autophagy flux and activate apoptosis in the pancreatic acinar cell, thus damaging rat pancreas.