| Objective: This study aims to explore the protective effect of kaempferol(KAE),an active monomer extracted from Penthorum chinense Pursh,on liver toxicity,oxidative stress,inflammatory response,and cell apoptosis.The study aims to investigate the underlying mechanism of KAE in alleviating hepatic ischemia/reperfusion injury(HI/RI)using molecular biology methods.By investigating the hepatic protective effect of KAE based on oxidative stress and inflammation,the study aims to expand the application of Penthorum chinense Pursh in treating liver diseases,and provide a therapeutic target and development platform for liver protection research.Methods: 1.Firstly,this study isolated and extracted high-purity flavonoid monomer KAE from Penthorum chinense Pursh using HPLC.Different doses of KAE were pre-treated in disease models constructed in mice,primary mouse liver cells,and normal human liver cell lines.2.Mice serum transaminase was detected.3.The contents of MDA,SOD,and GSH in the mice liver homogenate were measured.4.Primary mouse liver cells were isolated and extracted by in situ liver retrograde perfusion.5.The toxicity range of KAE pretreatment was determined by CCK-8 method,and the optimal concentration was selected.6.The effects of KAE pretreatment on the expression of inflammatory factors,apoptosis in vivo and in vitro,and Nrf2/HO-1 signaling pathway were detected by WB.7.H&E staining was used to analyze the morphological and structural changes of mice liver tissue,TUNEL staining was used to analyze mice liver cell apoptosis,and IHC staining was used to analyze the expression of downstream protein HO-1 of Nrf2.8.DCFH-DA fluorescent probe was used to detect and quantitatively analyze the generation of ROS in liver cells.Results: 1.KAE pretreatment exhibited a dose-dependent decrease in transaminase elevation induced by I/R,along with maintenance of relatively intact hepatic lobular architecture and smaller necrotic areas.2.The study confirmed the absence of significant toxicity of 60 mg/kg KAE dose in mice in the single drug group,and determined that the optimal concentration of KAE for in vitro experiments was approximately 5 μM,while the dose of KAE inducing toxicity in liver cells was around 20 μM.3.KAE pretreatment significantly alleviated I/R-induced oxidative stress,as evidenced by decreased production of the oxidative product MDA,increased levels of antioxidant enzymes SOD and GSH,and reduced generation of ROS induced by H/R in vitro.4.KAE inhibited the activation of NF-κB/p65,decreased the release of pro-inflammatory cytokines(TNF-α and IL-6),and increased the release of anti-inflammatory cytokine IL-10,thus attenuating the inflammatory response induced by I/R.5.KAE pretreatment downregulated the expression of apoptosis-related protein Bax and upregulated the expression of anti-apoptotic protein Bcl-2 both in vivo and in vitro,and significantly reduced liver cell apoptosis induced by I/R,as confirmed by TUNEL staining.6.KAE pretreatment upregulated the expression of proteins related to the Nrf2/HO-1 signaling pathway both in vivo and in vitro,and IHC staining of HO-1 indicated a significant increase in the number of positive cells after KAE pretreatment.Conclusion: 1.KAE pretreatment could inhibit inflammation and oxidative stress induced by I/R in the mouse model of HI/RI,thus further alleviating hepatocyte apoptosis.In the H/R model of liver cells,KAE pretreatment could significantly reduce the generation of ROS and alleviate hepatocyte apoptosis.2.The anti-inflammatory and anti-oxidative effects of KAE are related to the inhibition of NF-κB/p65 signaling pathway and activation of Nrf2/HO-1signaling pathway.3.This study further elucidates the specific mechanism of KAE pretreatment in alleviating HI/RI,suggesting that the active monomer KAE from Penthorum chinense Pursh is a potential candidate drug for the prevention and treatment of HI/RI. |
PDF Full Text Request