Effect and Mechanism of "Liver-Soothing and Spleen-Invigorating" Therapies Against Liver Injury in Rats： An Exploration Based on TGR5 and Intestinal Mucosal Barrier Function / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo explore the effects of different treatment methods of "soothing liver， invigorating spleen， soothing liver and invigorating spleen， soothing liver first and then soothing liver and invigorating spleen， as well as invigorating spleen first and then soothing liver and invigorating spleen" on liver depression combined with liver injury in rats and their action mechanisms. MethodA six-week rat model of liver depression combined with liver injury was established by restraint stress and subcutaneous injection of carbon tetrachloride （CCl4， 5.89 g·kg-1， once every three days）. At the same time， the drugs were given by gavage. Forty-eight male SD rats of clean grade were randomly divided into eight groups， namely the normal group， model group， bicyclol （0.2 g·kg-1） group， Sinisan （4.32 g·kg-1） group， Liu Junzitang （9.26 g·kg-1） group， Chaishao Liu Junzitang A （Chai A， soothing liver and invigorating spleen，13.57 g·kg-1） group， Chaishao Liu Junzitang B （Chai B， soothing liver first and then soothing liver and invigorating spleen， 13.57 g·kg-1） group， and Chaishao Liu Junzitang C （Chai C， invigorating spleen first and then soothing liver and invigorating spleen， 13.57 g·kg-1） group， with six rats in each group. The pathological changes in liver and colon tissues of each group were observed under light microscope and electron microscope. The serum biochemical indexes of the liver were detected using an automatic biochemical analyzer. The relative mRNA expression levels of Takeda G protein-coupled receptor 5 （TGR5） and intestinal mucosal zona occluden-1 （ZO-1）， Occludin， and Claudin-1 in the liver and colon were detected by reverse-transcription polymerase chain reaction （RT-PCR）. The positive expression rate of proliferating cell nuclear antigen （PCNA） in the colon was detected by immunohistochemistry. ResultCompared with normal group， the model group exhibited significantly elevated serum alkaline phosphatase （ALP）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， total bilirubin （TBIL）， and direct bilirubin （DBIL） （P<0.01）， lowered TGR5 mRNA expression in liver tissue， up-regulated TGR5 mRNA expression in the colon tissue （P<0.05，P<0.01）， and down-regulated ZO-1， Occludin， and tight junction protein-1 (Claudin-1) mRNA expression and PCNA in the colon tissue （P<0.01）. Compared with the model group， bicyclol and Chai C remarkably decreased the levels of serum ALP， ALT， AST， TBIL， and DBIL （P<0.05，P<0.01）， while Liu Junzitang， Chai A， Chai B， and Chai C significantly up-regulated the TGR5 mRNA expression in the liver and down-regulated its expression in the colon （P<0.01）. Bicyclol， Chai A， Chai B， and Chai C enhanced the ZO-1 and Claudin-1 mRNA expression in the colon （P<0.05，P<0.01）. Bicyclol， Sinisan， and Chai C increased PCNA expression （P<0.01）. The comparison with the Chai C group showed that the TGR5 mRNA expression in the liver and ZO-1 mRNA expression in the colon of the bicyclol and Sinisan groups were lower， whereas the TGR5 mRNA expression in the colon was higher （P<0.01）. However， the PCNA expression in the colon of the Liu Junzitang and Chai B groups declined significantly （P<0.05）. ConclusionIn the presence of liver injury， invigorating spleen first helps to relieve the liver injury， and the efficacy of "spleen-invigorating" therapy in increasing the intestinal mucosal tight junction proteins and improving the gastrointestinal function is related to its activation of TGR5 to improve the intestinal mucosal barrier function， promote the renewal of intestinal stem cells， and drive the regeneration after injury.