The Molecular Mechanism Of The Traditional Chinese Medicine Formulas In Preventing Acute Liver Injury

Acute liver injury（ALI）has emerged as a significant global public health concern,posing a serious threat to human health.Early symptoms of ALI are often subtle and easily overlooked;without timely treatment,it can progress to more severe liver conditions such as liver failure and fibrosis.Although several Western medications are available for treating ALI,their effectiveness is limited by side effects.Therefore,preventive measures and daily liver care are more practically significant than treatment alone.Compared to Western medicines,the traditional Chinese medicine（TCM）formulas are highly favored for their multi-component,multi-target approach and low toxicity,making them popular for both disease prevention and treatment.In this study,we used a carbon tetrachloride（CCl₄）-induced mouse model of ALI to investigate the preventive effects and molecular mechanisms of two TCM formulas:Yinchen sini decoction（YCSND）and Xiaobugan decoction（XBGD）.The TCM formula Hugan pill（HGP）was used as a positive control.The main findings were presented in the following four sections:1.Analysis of TCM formula components and network pharmacology prediction:We determined the active components and antioxidant activity in vitro of HGP,YCSND,and XBGD using chemical reagent methods.Ultra high performance liquid chromatography coupled with Q-Exactive focus mass spectrometry（UHPLC-QE-MS）was employed to identify the compounds in three TCM formulas.The identified active components were then subjected to network pharmacology prediction.The results indicated that YCSND contained the highest levels of polyphenols and flavonoids,while XBGD had the highest polysaccharide content.All three TCM formulas demonstrated good free radical scavenging activity in vitro.UHPLC-QE-MS identified100 compounds in HGP,89 in YCSND,and 78 in XBGD.Network pharmacology analysis based on these identified compounds revealed that the most significant pathway for ALI prevention by all three formulas was the phosphoinositide 3-kinase（PI3K）/protein kinase B（AKT）signaling pathway.2.Protective effects of TCM formulas on hepatocytes:Using a CCl₄-induced hepatocyte injury model,we evaluated cell viability with the MTT assay and measured the expression of relevant genes with reverse transcription quantitative polymerase chain reaction（RT-q PCR）.We also examined physiological indicators,mitochondrial membrane potential,and apoptosis to explore the protective effects of HGP,YCSND,and XBGD on liver cells.The analysis of physiological indicators showed that all three TCM formulas mitigated CCl₄-induced Hep G2 and L02 cell damage by inhibiting the increase of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in the cell supernatant,enhancing the activity of superoxide dismutase（SOD）and the content of glutathione（GSH）,reducing reactive oxygen species（ROS）production,improving mitochondrial membrane potential,and suppressing apoptosis.RT-q PCR results confirmed that all three TCM formulas alleviated CCl₄-induced hepatocyte damage by inhibiting the PI3K/AKT signaling pathway,reducing inflammatory responses,and downregulating the expression of pro-apoptotic genes.3.Preventive effects of TCM formulas on ALI in mice:Using a CCl₄-induced ALI mouse model,we investigated the preventive effects of TCM formulas through various methods including physiological indicator measurements,hematoxylin and eosin（H&E）staining,RT-q PCR,and western blotting（WB）.The results indicated that HGP,YCSND,and XBGD effectively reduced serum levels of liver-injury markers such as ALT,AST,alkaline phosphatase（ALP）,and total bilirubin（TBIL）.Additionally,these formulas decreased malondialdehyde（MDA）content in the liver and serum,increased superoxide dismutase（SOD）activity,and enhanced total antioxidant capacity（T-AOC）.HGP and XBGD significantly reduced serum total bile acids（TBA）levels,while HGP and YCSND effectively restored glutathione（GSH）levels.HGP and YCSND primarily inhibited inflammatory responses by downregulating the gene expression of nuclear factor kappa B（Nfκb）.XBGD reduced inflammation by increasing the level of the anti-inflammatory cytokine interleukin 10（Il10）at medium doses.All three TCM formulas effectively decreased the m RNA levels of pro-apoptotic genes Bcl2-associated X protein（Bax）and Caspase3.Compared to HGP and YCSND,XBGD significantly increased the level of the anti-apoptotic gene B-cell lymphoma 2（Bcl2）.The TCM formulas did not significantly affect cell autophagy,bile acid metabolism,or fatty acid synthesis gene expression.However,they improved fatty acid oxidation to some extent by increasing the m RNA levels of peroxisome proliferator-activated receptor alpha（Pparα）.Furthermore,it was confirmed at both m RNA and protein levels that all three TCM formulas effectively inhibited the PI3K-AKT signaling pathway.4.Effects of TCM formulas on host metabolism and gut microbiota in ALI Mice:Using high-throughput sequencing combined with proton nuclear magnetic resonance（¹H-NMR）,we explored the effects of HGP,YCSND,and XBGD on the microbiota of the small intestine and cecum,as well as on the metabolites in the liver and small intestine.The study revealed the following:（1）CCl₄ primarily disrupted the small intestine microbiota in ALI mice,with minimal impact on the cecum microbiota.HGP,YCSND,and XBGD effectively restored the relative abundance of Bifidobacterium,Bryobacter,Christensenellaceae＿R-7＿group,and[Ruminococcus]＿torques＿group in the small intestine.YCSND showed regulatory effects on Akkermansia,Butyricicoccus,Lachnospiraceae＿UCG-001,Methanosaeta,Streptococcus,Intestinimonas,and Ruminoccocaceae＿UCG-005,especially in the high-dose group.XBGD effectively restored the relative abundance of Aeriscardovia,Acinetobacter,Intestinimonas,Methanosaeta,and Megamonas in the small intestine.（2）For the cecum microbiota,the CCl₄-upregulated Ruminococcaceae＿NK4A214＿group was restored by HGP,YCSND,and XBGD.All three TCM formulas effectively suppressed some potential pathogenic bacteria in the cecum.（3）RT-q PCR results indicated that HGP,YCSND,and XBGD effectively restored the m RNA levels of ileum tight junction protein zonula occludens 1（Zo1）,regenerating islet-derived protein III（Reg3g）,tumor necrosis factor-alpha（Tnfα）,and monocyte chemoattractant protein 1（Mcp1）,thus improving intestinal homeostasis in ALI mice.Additionally,XBGD increased the m RNA levels of ileum Il10,occludin,and mucin 2（Muc2）,showing a stronger regulatory effect on the intestinal barrier compared to HGP and YCSND.（4）¹H-NMR metabolite analysis revealed that CCl₄ significantly disrupted liver metabolism and,to a certain extent,affected small intestine metabolism.The disrupted liver metabolites included those involved in amino acid metabolism（glutamate,N-acetylglutamate,taurine）,energy metabolism（glucose,acetate,lactate,3-hydroxybutyric acid,fumarate,malic acid,succinate）,as well as betaine,choline,glycerol,and trimethylamine N-oxide.Disrupted small intestine metabolites included acetoacetate,acetate,citrate,ethanol,and trimethylamine.HGP,YCSND,and XBGD partially restored liver and intestinal metabolism,with XBGD at medium doses showing a stronger regulatory effect on liver metabolites.Spearman correlation analysis indicated that the small intestine microbiota regulated by HGP,YCSND,and XBGD were significantly correlated with ALI-related serum markers,liver pro-inflammatory cytokines,and antioxidant capacity.The cecum microbiota regulated by the three TCM formulas showed a weaker correlation with liver injury-related indicators.Liver metabolites such as choline,glycerol,taurine,and trimethylamine N-oxide were positively correlated with the small intestine microbiota regulated by the three TCM formulas.In conclusion,this study conducted a comprehensive investigation into the molecular mechanisms underlying the prevention of acute lung injury（ALI）by three traditional Chinese medicine formulations,building upon UHPLC-QE-MS predictive analysis combined with network pharmacology.Through a systematic approach involving the analysis of physiological and biochemical indicators,gene expression levels,metabolomics,and gut microbiota,both at cellular and animal levels,the research elucidated that HGP,YCSND,and XBGD exhibit preventive effects against ALI.The findings indicate that these formulations exert their effects by modulating the PI3K/AKT signaling pathway,reducing inflammation,inhibiting hepatocyte apoptosis,enhancing antioxidant capacity,regulating intestinal microbiota,improving intestinal homeostasis,and restoring liver metabolism.Of particular significance is the robust regulatory capacity of the edible formulation XBGD on liver metabolism and intestinal barrier function.This study not only establishes a theoretical framework for the preventive efficacy of traditional Chinese medicine formulations against ALI but also offers novel insights into their molecular mechanisms.