| Background: Pancreatic ductal adenocarcinoma(PDAC)is one of the most aggressive malignancies in the world.Extreme hypoxia in the tumor core of advanced PDAC causes abnormal transmission of electronic respiratory chains in the inner mitochondrial membrane,promoting a more aggressive and metastatic phenotype.The high expression of HIF-1,a key target,mediates the occurrence of a variety of malignant biological behaviors.The protein deacetylase SIRT3 performs a unique function by mediating interactions between mitochondria and intracellular signaling.In recent years,traditional Chinese medicine ingredients such as natural products have been found to be used in combination with chemotherapy drugs to reduce the side effects of the latter and improve prognosis.Proanthocyanidins is a class of polyphenolic compounds widely present in the plant kingdom and is not genotoxic.Numerous in vivo and in vitro experiments have shown that proanthocyanidins achieves anti-tumor effects by inhibiting a variety of molecular targets,but the molecular mechanism of targeting pancreatic cancer signaling pathways has not been clarified.Therefore,investigating the molecular mechanism of proanthocyanidins against tumor will help the development of new clinical drugs and provide experimental basis for their application in the treatment of pancreatic cancer.Objective: Low oxygen state and high glucose metabolism are common environmental conditions for tumor cell survival,while PDAC cells show unique adaptability to extreme hypoxia.Therefore,this study combined network pharmacology and bioinformatics to investigate the relevant role and mechanism of SIRT3/HIF-1α signaling pathway in proanthocyanidin inhibiting the proliferation of PANC-1 cells in hypoxic pancreatic cancer under the premise of HIF-1α high expression.Methods: 1.Search for proanthocyanidins,pancreatic cancer,oxidative stress and other potential targets through Super-PRED,Pub Chem and Dis Ge NET databases,and Venny software was used to obtain the joint targets of proanthocyanidins in regulating the level of oxidative stress in pancreatic cancer.2.Use String database and Cytoscape to investigate the target gene PPI network and generate the main core target network.3.The expression and correlation between SIRT3 and HIF-1α gene in pancreatic cancer were predicted through the GEPIA website.4.The anaerobic culture bag physical induction method was used to simulate the hypoxic environment in PANC-1 cells.According to the purpose of the study,it was divided into normoxic control group,hypoxic model group and low(15μg/ml)and medium(30μg/ml),high(60μg/ml)concentration group of proanthocyanidins.5.The effects of different concentrations of proanthocyanidins on the viability of PANC-1 cells at 24 h and 48 h were detected by CCK-8 method under normal oxygen and hypoxic conditions,respectively,and the median inhibitory concentrations IC50 were calculated to determine the final administration concentrations.6.The apoptosis level of PANC-1cells was detected by Annexin V-FITC/PI double staining method,and the level of intracellular reactive oxygen species(ROS)was detected by DCFH-DA fluorescent probe.Biochemical kits were used to detect the intracellular superoxide dismutase(SOD)activity and total antioxidant capacity(T-AOC).7.Western Blot was used to detect the changes in the expression of SIRT3 and HIF-1α proteins in pancreatic cancer cells.The mechanism of SIRT3/HIF-1α signaling pathway in the influence of proanthocyanidins on the biological activity of hypoxic pancreatic cancer cells was preliminarily revealed.Result: 1.A total of 166 proanthocyanidin-related targets,362 human pancreatic cancer-related targets,407 oxidative stress-related targets and 406 aging-related targets were collected through Dis Ge NET databases.2.Venny and Cytoscape software analyzed 17 core targets of proanthocyanidins in regulating oxidative stress in pancreatic cancer,among which the key targets involved SIRT3 and HIF-1α.3.The GEPIA database predicted that there was no significant difference in the expression of SIRT3 in pancreatic cancer and normal tissues,but there was still an up-regulation trend(P(29)0.05),while the expression of HIF-1α was significantly upregulated and SIRT3 was positively correlated with HIF-1α(P(27)0.05).4.Under normoxic condition,CCK-8 experiments showed that proanthocyanidins could significantly inhibit the viability of PANC-1 cells(P(27)0.05).The median inhibitory concentrations IC50 at 24 h and 48 h were 50.85 μg/ml and 35.25 μg/ml,respectively.Under hypoxic condition,higher gradient proanthocyanidins significantly inhibited the viability of PANC-1cells(P(27)0.05),and the median inhibitory concentration IC50 at 24 h and 48 h were81.28 μg/ml and 44.95 μg/ml,respectively.5.In situ fluorescence detection showed that compared with the normoxic control group,the intracellular ROS level in the hypoxic model group was significantly increased and no obvious apoptosis was seen(P(27)0.001).Compared to the hypoxic model group,proanthocyanidins can reverse intracellular ROS levels and induce apoptosis(P(27)0.05 or P(27)0.001).Antioxidant activity assay showed that compared with the normoxic control group,the intracellular SOD activity decreased significantly and the T-AOC increased in the hypoxic model group(P(27)0.05).Compared to the hypoxic model group,proanthocyanidins increased the intracellular SOD activity and T-AOC in a concentration-dependent manner(P(27)0.05 or P(27)0.001).6.The results of Western Blot showed that compared with the normoxic control group,there was no significant change in SIRT3 expression in the hypoxic model group(P(29)0.05),and the expression of HIF-1α was significantly upregulated(P(27)0.001).Compared with the hypoxic model group,proanthocyanidins could significantly downregulate the expression of SIRT3 and HIF-1α proteins in PANC-1 cells(P(27)0.05 or P(27)0.001).Conclusion: 1.Based on network pharmacology and bioinformatics analysis,the expression of SIRT3 and HIF-1α genes in pancreatic cancer cells was positively correlated,and the higher expression levels of the two may be related to the higher redox levels in pancreatic cancer cells.2.This study confirmed that proanthocyanidins can inhibit the proliferative activity of the pancreatic cancer cell line PANC-1,which can be weakened by hypoxic conditions,and the mechanism may be related to the inhibition of the ROS-mediated SIRT3/HIF-1α signaling pathway. |
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