Hesperetin Derivative Inhibits CCl₄-induced Mice Acute Liver Injury By Targeting PTP1B

Acute liver injury（ALI）is an acute inflammatory disease of the liver caused by various etiologies,including liver injury caused by hepatitis,drugs and toxins.Acute liver injury is characterized by sudden liver dysfunction and inflammatory response.At present,the treatment of acute liver failure is still an extremely important and challenging problem in clinical medicine.Related studies have shown that resident macrophages called Kupffer cells（KCS）play a crucial role in the initial stress response during liver injury.Activated Kupffer cells（KCS）are widely recognized as one of the major cell types leading to ALI,releasing various metabolites that cause cell damage,including superoxide radicals,nitric oxide,proteases,and proinflammatory cytokines.Thus,regulating macrophage activity provides an excellent therapeutic strategy to alleviate severe liver inflammation.Previous studies in our laboratory show that,compound 14（HD-14）of the hesperitin derivative produces proinflammatory cytokines by inhibiting macrophages,It has direct inhibitory effect on acute liver injury.Hesperitin is a flavanone glycosides,Natural in the pericarp of citrus plants.Previous studies have shown that,There are many pharmacological effects of hesperitin,including anti-inflammatory,anti-tumor,antioxidant and neuroprotective effects.The experiment found,The compound 2（HD-2）,which was synthesized by structural modification of hesperitin in our laboratory,attenuated the inflammatory response of LPS treated RAW264.7 cells,The experiment also found that,HD-2 can promote PTP1B expression.Protein tyrosine phosphatase（PTPs）belongs to a superfamily.It can dephosphorylate tyrosine on its protein substrate,play a key role in many biological processes.Protein tyrosine phosphatase 1 B（protein tyrosine phosphatase 1 B,PTP1B）is an intracellular PTPs,involved in the regulation of many cellular signaling molecules,It has many regulatory effects in liver regeneration,hepatocyte apoptosis and liver cancer.Given the regulatory role of PTP1B in cells,suggesting that HD-2 anti-acute liver injury effects may be achieved by PTP1B.The above contents have not been reported in the literature,Therefore,this experiment focuses on the therapeutic effect and partial mechanism of hesperitin derivatives on acute liver injury induced by carbon tetrachloride in mice.This experiment adopts the method of combining in vivo experiment with in vitro experiment.In vivo experiments:8-10 weeks C57BL/6J mice were divided randomly into seven groups（n=10 per group）:Control group,CCl₄group,HD-2 treatment group（50 mg/kg,100 mg/kg,200 mg/kg）,and Silymarin group（100 mg/kg）,normal plus HD-2 treatment group（200 mg/kg）.HD-2 treatment group,CCl₄group and Silymarin group were injected intraperitoneally with a single dose of 1%CCl₄（10 ml/kg in oil）to induce acute liver injury model,besides,mice in HD-2 treatment group and Silymarin group were given via gavage different concentrations of HD-2 and Silymarin for 7 days before.Control groups were injected intraperitoneally the same volume of vehicle（oil）.Normal plus HD-2 treatment group（200 mg/kg）were only given via gavage high concentrations of HD-2 for 7 days.The normal control group was intraperitoneally injected with the same volume of olive oil.To determine the extent of liver damage in each group,Histopathological examination of liver tissue with HE staining and immunohistochemistry;ALT and AST、TG and T-CHO、T-BIL kit to detect liver injury index level;The protein and m RNA expression levels of TNF-α、IL-1β、IL-6 in liver tissue of acute liver injury mice were detected by Western blot and RT-q PCR.The results show,HD-2 has certain therapeutic effect on CCl₄induced acute liver injury in mice.In vitro experiments:First,the effect of different concentrations of HD-2 on the cell viability of RAW264.7 cells was detected by MTT method.To determine the cytotoxicity of HD-2 to RAW264.7 cells;Reusing LPS and different concentrations of HD-2 cells for 48 h,MTT method was used to screen the minimum concentration of nontoxic to cells.MTT methods were used to detect the effects of HD-2 on RAW264.7cells.Then transfected si RNA and p EX3-PTP1B;After 24 h of culture,Extracting Protein and Total RNA,WB test the protein expression of PTP1B and inflammatory factors,The gene expression of PTP1B and inflammatory factors was detected by RT-q PCR experiment.The final results show that,HD-2 can significantly inhibit RAW264.7 activation and proliferation.To sum up,the HD-2 is to down-regulate the expression of PTP1B,and ultimately affect acute liver injury.Hence,HD-2 can be used as a potential anti-inflammatory monomer compound for the treatment of acute liver injury.