Baicalin Alleviates AFB1-induced Hepatic Cell Apoptosis And Its Mechanism

Aflatoxin is known to be the most toxic of all mycotoxins,and it has been found to be carcinogenic,teratogenic,and mutagenic.Out of the 21 different types of aflatoxins that have been identified,Aflatoxin B1(AFB1)is the most harmful to both humans and animals.Unfortunately,AFB1 is commonly found in animal feed,and it is very difficult to avoid.This can lead to liver dysfunction and reduced productivity in livestock and poultry,resulting in significant economic losses for the livestock industry.It is crucial to discover methods to prevent and decrease AFB1 contamination in order to promote the growth of the livestock and poultry industry.Baicalin is a flavonoid glycoside that serves as the principal active ingredient in Scutellaria baicalensis.It possesses a range of pharmacological effects,including antiinflammatory,antibacterial,and anti-tumor properties.Baicalin’s protective effects on liver disease have gained significant attention in recent years.Multiple studies have demonstrated that baicalin can mitigate liver injury caused by apoptosis by regulating the expression of apoptotic genes,reducing lipid peroxidation,increasing the activity of antioxidant enzymes,and preserving the structure and function of mitochondria.However,the specific targets and mechanisms of baicalin’s action in alleviating AFB1-induced apoptosis in hepatocytes have not been thoroughly investigated.This project utilized network pharmacological analysis and molecular docking technology to identify the key targets of baicalin in reducing AFB1-induced apoptosis in hepatocytes.An in vitro apoptosis model was constructed using L02 cells,which were incubated with varying doses of baicalin.The purpose of this study is to examine the molecular mechanism of AFB1-induced apoptosis in hepatocytes by focusing on the core targets and their related pathways.Additionally,we will investigate the anti-apoptotic targets of baicalin and explore its mechanism of action in alleviating AFB1-induced liver injury.The results of this study will provide a scientific basis and new ideas for preventing and controlling AFB1 hazards.The results of the study are as follows:1.The potential targets of baicalin in alleviating AFB1-induced liver cell damage and their roles in gene function and signal transduction were determined in this study.Network pharmacology combined with molecular docking techniques were used to identify the core targets of baicalin in alleviating AFB1-induced liver cell apoptosis.The results showed that the alleviation of AFB1-induced liver damage by baicalin may be related to the targets JNK,GPT,TLR4,HPRT1,CYP1A1,HMOX1,Bax,TP53,PTGS2,caspase 3,MAPK1,EGFR,TGFB1,and CYP1A2,among which JNK,Bax,and caspase 3 were identified as key targets.GO enrichment analysis and KEGG pathway analysis results showed that the potential key targets may induce liver cell apoptosis by mediating the MAPK apoptosis signaling pathway,leading to oxidative stress and mitochondrial damage.Molecular docking results showed that baicalin has binding potential with the potential core target JNK,indicating that JNK may play an important role in baicalin’s alleviation of AFB1-induced liver cell apoptosis,and baicalin has the potential to target JNK.2.Baicalin can effectively alleviate AFB1-induced liver cell apoptosis.In this study,the AFB1 concentration for in vitro modeling was determined by cell viability assay(CCK8)combined with protein immunoblotting.The cell toxicity results showed that AFB1 significantly inhibited cell growth at concentrations of 5-40 μg/m L,and the protein immunoblotting results showed that AFB1 induced the most significant liver cell apoptosis at a concentration of 10 μg/m L.Therefore,subsequent experiments induced liver cell apoptosis using 10 μg/m L AFB1.Secondly,the cell toxicity of baicalin on L02 was detected by CCK8 assay,and the results showed that baicalin at concentrations of 0-100 μg/m L had no cell toxicity on L02 cells.Therefore,baicalin concentrations of 25,50,and 100 μg/m L were selected as treatment concentrations for subsequent experiments.Finally,an in vitro AFB1-induced liver cell apoptosis model was constructed,and different concentrations of baicalin were used for intervention.The results showed that baicalin significantly alleviated AFB1-induced liver cell apoptosis,cell cycle arrest,and expression of apoptosis-related genes.3.Baicalin targets JNK to alleviate AFB1-induced hepatocyte apoptosis.In this study,JNK-specific inhibitor SP600125 was used to simulate the inhibition of JNK protein activity by baicalin in cells.The pc DNA3.0-JNK overexpression vector was transfected into cells to simulate the enhanced JNK protein activity in L02 cells induced by AFB1.The role of the core target JNK in baicalin’s alleviation of AFB1-induced hepatocyte apoptosis was determined.The results showed that baicalin alleviated AFB1-induced hepatocyte apoptosis by inhibiting JNK protein activity.Nuclear-cytoplasmic separation and immunofluorescence experiments showed that AFB1 enhanced the expression levels of JNK in the nucleus and cytoplasm,respectively,activating the JNK/AP-1 signaling axis,JNK/Bcl2/Bax/caspase 3signaling pathway,and enhancing the cleaved caspase 3-mediated cleavage of PARP1,inducing hepatocyte apoptosis.Baicalin inhibited the activation of the apoptotic pathway induced by AFB1.In addition,the drug affinity responsive target stability(DARTS)technique showed that baicalin could target and bind to JNK.The above study showed that JNK could mediate AFB1-induced hepatocyte apoptosis,and AFB1 could induce hepatocyte apoptosis by phosphorylating JNK and activating the downstream molecules of the death receptor pathway(JNK/AP-1)and the mitochondrial-dependent apoptosis pathway(JNK/Bcl2/Bax/caspase 3/PARP1).Therefore,this study revealed that JNK was activated by AFB1,inducing hepatocyte apoptosis through the cross-talk and synergy of multiple pathways.JNK may be the core target for treating AFB1-induced liver damage.Baicalin targets JNK,inhibits JNK phosphorylation,and alleviates AFB1-induced hepatocyte apoptosis..In summary,this study investigated the potential of baicalin to target JNK through network pharmacology and molecular docking.The initial hypothesis was that JNK plays a crucial role in baicalin’s ability to alleviate AFB1-induced apoptosis in hepatocytes.Flow cytometry was then used to detect apoptosis and cell cycle changes,which showed that baicalin significantly inhibited the rate of AFB1-induced apoptosis and cell cycle arrest in hepatocytes,indicating that baicalin could effectively alleviate AFB1-induced apoptosis in hepatocytes.Furthermore,AFB1 triggers apoptosis in hepatocytes through various pathways.It phosphorylates JNK,which activates the death receptor pathway(JNK/AP-1),the mitochondria-dependent apoptotic pathway(JNK/Bcl2/Bax/caspase 3/PARP1).The DARTS technique conclusively demonstrated that baicalin targets JNK binding,inhibits JNK protein activity,and effectively alleviates AFB1-induced apoptosis in hepatocytes.This study provides a clear understanding of the molecular mechanism underlying the effect of baicalin in preventing AFB1-induced hepatocyte apoptosis.Furthermore,this study provides a scientific basis for the use of baicalin as a potential treatment for AFB1-induced liver injury in livestock.