Ginsenoside Rb1 Alleviates Liver Injury Induced By 3-chloro-1,2-propanediol Through Stimulating Autophagic Flux Via MiR-128

Liver is the most important metabolic and detoxification organ of the human body.In addition to the metabolism of nutrients,liver is also an important site for the biotransformation of many non-nutritional substances(drugs,poisons and toxic metabolites),so it is also a common target organ for toxic effects of toxic substances.Liver injury refers to the damage of hepatocytes caused by a variety of pathogenic factors invading the liver,resulting in impaired normal physiological functions of liver,which can lead to a series of symptoms and even death.3-chloro-1,2-propanediol(3-MCPD)is a well-known contaminant that was produced in the thermal processing of food and has the characteristics of high pollution and strong toxicity.Studies have shown that the liver is the acute toxic target organ of 3-MCPD,and exposure to 3-MCPD can lead to a variety of pathological changes in hepatocytes.Ginseng is a traditional Chinese herbal medicine in China.Ginsenoside Rb1(Gs-Rb1)is the most abundant component in ginsenoside and has a variety of biological activities.Studies have proved that Gs-Rb1 has a certain alleviating effect on liver injury,but the specific mechanism is still unclear.As a cellular defense and stress regulation strategy,autophagy is involved in the pathogenesis of a variety of liver diseases.Therefore,autophagy is likely to be one of the mechanisms of drug protection against liver injury.In this study,autophagy was taken as the entry point to study the hepatotoxicity mechanism of 3-MCPD and the alleviating effect of Gs-Rb1 on hepatotoxicity induced by 3-MCPD.The main research contents and results are as follows:(1)3-MCPD inhibits autophagic flux by impairment of lysosomal function in HepG2 cells.Western blot was used to detect the expression of microtubule-associated protein light chain 3(LC3)under different concentrations of 3-MCPD treatment for different periods of time,and the number of autophagosomes in HepG2 cells was observed by MDC staining and transmission electron microscopy.It was confirmed that 3-MCPD could cause the accumulation of autophagosomes in HepG2 cells.Two autophagy inhibitors,chloroquine(CQ)and 3-methyladenine(3-MA),were added in the experimental process.The expressions of LC3 protein and P62/SQSTM1 protein were detected by Western blot,and the fluorescence expression after transfection of mRFP-GFP-LC3 autophagy double-labeled adenovirus was observed by confocal laser scanning microscopy.It was confirmed that the main reason of 3-MCPD-induced intracellular autophagosome accumulation was the inhibition of lysosomal degradation.The co-localization of LC3 and LAMP1 in cells after immunofluorescence staining was observed by confocal laser scanning microscopy,and it was confirmed that 3-MCPD had no inhibitory effect on the binding of autophagosome and lysosome.The effect of3-MCPD on lysosome pH was observed by Lyso-Tracker Red probe staining(LTR)and Acridine orange(AO)staining.It was confirmed that 3-MCPD led to an increase in lysosomal pH and alkalinization of lysosomes in HepG2 cells.The expression of TFEB protein in cytoplasm and nucleus was detected by Western blot,and the mRNA expression of TFEB and its target genes was detected by qPCR.It was confirmed that3-MCPD inhibited the nuclear expression and transcription of TFEB,and then inhibited the expression of lysosomal related target genes.TFEB was overexpressed by adenovirus transfection,LC3 protein and P62/SQSTM1 protein were detected by Western blot,and lysosomal pH was observed by LTR probe.It was confirmed that exogenous TFEB could rescue 3-MCPD-induced autophagic flux blockage and lysosomal alkalization.(2)Gs-Rb1 alleviates 3-MCPD-induced liver injury in ICR mouse by stimulating autophagic flux.By drawing the survival curve,calculating the weight change and liver index of mice,observing the liver histopathological section and detecting the markers of liver injury(ALT and AST),it was confirmed that 3-MCPD-induced liver injury in ICR mouse,and the liver injury could be alleviated by Gs-Rb1.The expressions of LC3 protein and P62/SQSTM1 protein of liver tissue in ICR mouse were detected by Western blot,and it was confirmed that Gs-Rb1 could restore autophagic flux blockage induced by 3-MCPD.By adding autophagy inhibitor CQ,it was found that when the stimulating effect of Gs-Rb1 on autophagic flux was inhibited,the liver injury induced by 3-MCPD reappeared,indicating that Gs-Rb1 can alleviate the liver injury induced by 3-MCPD in ICR mouse by stimulating autophagic flux.(3)Gs-Rb1 alleviates liver injury induced by 3-MCPD through stimulating autophagic flux via miR-128.The viability of HepG2 cells was detected by MTT assay,apoptosis rate was detected by flow cytometry(Annexin V/ PI double staining),and morphology of apoptotic cells was observed by DAPI staining.It was confirmed that3-MCPD could inhibit cell viability and promote apoptosis,and Gs-Rb1 could alleviate this toxic effect.The expressions of LC3 protein and P62/SQSTM1 protein were detected by Western blot,and the fluorescence expression after transfection of mRFPGFP-LC3 autophagy double-labeled adenovirus was observed by confocal laser scanning microscopy.It was confirmed that Gs-Rb1 could enhance autophagy and alleviate the autophagic flux blockage induced by 3-MCPD.The expression of TFEB protein in cytoplasm and nucleus was detected by Western blot,it was confirmed that Gs-Rb1 could alleviate the inhibition of TFEB nuclear expression induced by 3-MCPD.The mRNA expression of TFEB and its target gene and the expression of miR-128 were detected by qPCR,it was confirmed that Gs-Rb1 alleviated the inhibition of TFEB and its target gene mRNA expression induced by 3-MCPD and inhibited the increase of miR-128 expression induced by 3-MCPD.After transfection with hsa-miR-128 inhibitor and mimic,the expression of LC3 protein and P62/SQSTM1 protein were detected by Western blot,and apoptosis rate was detected by flow cytometry(Annexin V/ PI double staining).It was verified that miR-128 is a key target for Gs-Rb1 to alleviate the autophagic flux blockage induced by 3-MCPD and thus inhibit apoptosis.In summary,this study elucidated the molecular mechanism of 3-MCPD-induced autophagic flux blockage and the mechanism of Gs-Rb1 alleviating 3-MCPD-induced autophagic liver injury at the molecular levels(protein and mRNA).It was proved that miR-128 is the target of Gs-Rb1 to alleviating autophagic flux blockage caused by 3-MCPD disruption of lysosome function.Finally,it was confirmed that Gs-Rb1 could inhibit lysosomal dysfunction induced by 3-MCPD through miR-128-TFEB pathway,thereby enhance autophagic flux and alleviate liver injury.This study provides a reliable theoretical basis and target for the use of natural substances to reduce the harm of food processing pollutants on the human body,and has important theoretical and practical significance.