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Infantile cholestatic hepatopathy (ICH) is a clinical syndrome characterized by the accumulation of cytotoxic bile acids in infancy, leading to serious liver cirrhosis or liver failure. The aetiology of ICH is complicated and some of them is unknown. Regardless of the aetiology, the finial pathology of ICH is hepatocyte apoptosis caused by severe and persistent cholestasis. It is already known that activation of calcium-sensing receptor (CaSR) could lead to the apoptosis of cardiomyocytes. However, the mechanism by CaSR-mediated cholestasis-related hepatocyte apoptosis is not fully understood. Li-Dan-He-Ji (LDHJ), a Traditional Chinese Medicine prescription, was developed to treat ICH. Another aim of this study was to investigate the possible mechanisms of LDHJ in cholestasis-related hepatocyte apoptosis. Using the primary hepatocytes, we first investigated the molecular mechanism of CaSR-mediated hepatocyte apoptosis in cholestasis. Then we prepared LDHJ granules and used ultra-high-performance liquid chromatography to identify the predominant drugs; confirmed the stability of the main substances; and for cell experiments screened forsythoside-A, emodin and chlorogenic acid as the three active substances of LDHJ granules. In the young rats with ANIT-induced intrahepatic cholestasis and the primary hepatocytes with TCDC-induced cholestasis-related hepatocyte apoptosis, the levels of liver injury and cholestasis-related biomarkers, calcium-sensing receptor (CaSR), hepatocyte apoptosis, Bax/Bcl-2 ratio, Cytochrome-C, caspase-3, phosphorylated-c-Jun NH2-terminal kinase (p-JNK)/JNK, and p-P38/P38 were all increased, while the levels of p-extracellular signal-regulated kinase (p-ERK)/ERK were decreased. However, LDHJ granules and its three active substances effectively reversed these changes. Furthermore, the three active substances reduced the increases in the intracellular calcium concentration ([Ca2+]i) and ROS levels and attenuated the dissipation of the mitochondria membrane potential in the TCDC-induced primary hepatocytes. The opposite results were obtained from the TCDC-induced primary hepatocytes treated with an agonist of CaSR (GdCl3) plus forsythoside-A, emodin or chlorogenic acid. Based on the results from in vivo and in vitro studies, LDHJ functions as an antagonist of CaSR to regulate hepatocyte apoptosis in cholestasis through the mitochondrial pathway and mitogen-activated protein kinase pathway.

AimBile salt export pump (BSEP) have been confirmed to play an important role for bile acid canalicular export in the treatment of cholestasis. In this study, we investigated the stimulatory effect of emodin on BSEP signaling pathway in cholestasis.MethodsCell and animal experiments were given different concentrations of emodin. The BSEP upstream molecule farnesoid X receptor was down-regulated by small interfering RNA (siRNA) technology or guggulsterones and up-regulated by lentivirus or GW4064. Real-time PCR and Western blotting was employed to detect the mRNA and protein levels of BSEP in LO2 cell, rat primary hepatocytes and liver tissue. Immunohistochemistry (IHC) was used to examine the expression of BSEP in liver tissues. Rat liver function and pathological changes of liver tissue were performed by biochemical test and hematoxylin and eosin (HE) staining.ResultsEmodin could increase the mRNA and protein expression of BSEP and FXR. When down-regulating farnesoid X receptor expression with the siRNA or inhibitor guggulsterones, and up-regulating farnesoid X receptor expression with the lentivirus or agonist GW4064, emodin could increase the mRNA level of BSEP and FXR and the protein level of BSEP, FXR1, and FXR2. Emodin also had a notable effect on rat primary hepatocytes experiment, rat pathological manifestation, BSEP, FXR1, and FXR2 positive staining in liver tissues and the test of liver function.ConclusionEmodin has a protective effect and a rescue activity on cholestasis via stimulating FXR/BSEP pathways in promoting the canalicular export of accumulated bile.