| Background and objectivesNon-alcoholic fatty liver disease (NAFLD)encompasses a spectrum ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. Because it is closely associated with metabolic syndrome and insulin resistance, NAFLD currently is recognized as the most form of chronic liver disease and the leading cause of elevated liver function test in many parts of the world. NAFLD can coexist with viral hepatitis, and closely relate to type 2 diabetes, metabolic syndrome and cardiovascular factors. Therefore, NAFLD is a relevant challenge for liver units.The family of nuclear receptors , homologous steroid hormone receptors are ligand-dependent transcription factors, including the classic steroid hormone receptors and thymic hormone receptors, as well as many orphan nuclear receptors whose physiological ligands or activators have not found. They combine together the DNA sequence (response element) in transcription factor gene promoters of the target genes and activate them. The metabolic nuclear receptor superfamily are closely related to glycometabolism , lipometabolism and energy metabolism .They play a key role in many important life processes including glucose and lipid metabolism, insulin signal transduction, oxidative stress, inflammation, cell growth and differentiation, so on. As transcription factors, they strictly control the expression of their target genes, including many enzymes involved in glucose and lipid oxidation-reductive reaction, as well as the major intra-cellular antioxidant system. That they malfunction is a common pathophysiological basis which closely relate to a variety of metabolic diseases. Because many studies have shown that some of metabolic nuclear receptors, such as FXR, LXR and PPARa, are the regulation center of glucose and lipid metabolism in the body, they have become the focus of researching non-alcoholic fatty liver disease today. It is very important to elucidate the pathogenesis and find effective therapeutic target of NAFL that research these nuclear receptors ligands and their signal pathways.The farnesoid X receptor(FXR)is a ligand-activated transcription factor and a member of the nuclear recepor super family. FXR is activated by bile acids(BA) and expressed in liver, intestine, kidney and the adrenal gland. The FXR regulates BA synthesis from cholesterol by controlling the expression of key enzymes in the pathway, such as Cyp7a1(cholesterol-7?-hydroxylase), the rate-limiting enzyme of BA synthesis. Currently the FXR has been shown to serve as a metabolic receptor that can integrate metabolic and hormonal signals to changes in gene expression. As such, the FXR has a crucial role in the regulation of the hepatic metabolism of BA, lipids and glucose homeostasis. Abnormal expression of FXR is closely related with NAFL, gallstone disease(GD), and so on. There are few reports on FXR in human body. In this experiment, through investigating FXR expression and alteration of BA and triglyeride metabolism in the liver of NAFL with GD patients, GD patients and health adults, explores the role of FXR in the pathogenesis of NAFL and finds the novel therapeutic target for NAFL. MethodsTwenty patients who were operated cholecystectomy for gallstones gave their informed consent to participate in this study. They were divided into two groups. One group was named NAFL and GD group (NAFL+GD group) which composed of 10 patients with non-alcoholic fatty liver proved by histopathology. The other group was named GD group which composed of 10 patients proved by operation. Five donors liver transplantation served as controls constitute N group. All patients had been excluded viral, drug-induced, alcoholic and autoimmune liver disease, liver cancer, also ruled out tuberculosis, cancer and other systemic diseases. Hepatic tissues were obtained from the left edge of liver about 1 cm diameter size during operation and immediately placed -80?preservation. The degree of hepatic steatosis was evaluated by using HE stain. The expression of FXR, BSEP, and SREBP-1c in liver tissues was observed with immunohistochemical staining. The FAS-positive liver cells were visualized with laser scanning confocal microscopy. The expression of FXR, LXR, SREBP-1c, SHP and CYP7A1 protein was detected by using Western blot. The expression of FXR, LXR and SHP mRNA was analyzed with Real-Time PCR mRNA Quantification.This study was approved by the Ethics Committee at the second clinical institute of Chongqing Medical University.Results1. Immunohistochemical staining showed that expression of FXR was no difference among the three groups (P>0.05). Compared with the N group, expression of BSEP was decreased in the NAFL+GD group and the GD group(P<0.05), but there was no difference between the NAFL+GD group and the GD group (P>0.05). In the NAFL+GD group, expression of SREBP-1C was significantly increased compared with the GD group and N group (P<0.01), and closely related with the degree of hepatic steatosis.2. Immunofluorescence and confocal laser microscopy showed that the expression of FAS in NAFL+GD group was markedly stronger than that in GD group and N group.3. Western blot analysis showed that the expression of FXR protein was decreased in NAFL+GD group compared with the GD group and N group (P <0.05), while expression of LXR and SREBP-1C protein was increased (P<0.05). There were no differences both in expression of SHP and CYP7A1 protein among NAFL+GD group, GD group and N group (P> 0.05).4. Real-Time PCR mRNA Quantification showed that compared with the GD group and N group, expression of FXR mRNA was significantly reduced in the NAFL+GD group (P<0.01), expression of LXR mRNA was dramatically raised (P<0.01). No difference could be seen in SHP mRNA expression among three groups (P> 0.05).ConclutionsThese results provided molecular evidence for a relationship among expression of hepatic FXR, NAFL and GD in humans. The SREBP-1c transcription is induced by LXR(liver X receptor), SREBP-1c (Sterol response element-binding protein-1c ) control the expression of key lipogenic genes such as FAS, while BSEP(Bile salt exporting pump)plays an important role in the metabolism of bile acid. The FXR down regulates LXR, SREBP-1c expression and up regulates BSEP expression, therefore, a decreased expression of hepatic FXR could be contribute to the increased in hepatic triglyceride synthesis and cholesterol crystallization in NAFL patients. The FXR is a promising therapeutic target for treating or preventing NAFL by reduction of liver SREBP-1c expression and activity and attenuation of FAS expression and liver lipogenesis.Our studies did not demonstrate the critical dependence of the regulation of SREBP-1c or CYP7A1 by FXR on SHP in humans. |
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