ABSTRACT
OBJECTIVE Baicalin is a major flavonoid component of Scutellaria baicalensis, and has been used in the treatment of liver diseases for many years. However, the role of baicalin in estrogen-induced cholestasis (EIC) remains to be elucidated. This present study explored the protective effect of baicalin against estrogen-induced liver injury and further elucidated the mechanisms involved both in vivo and in vitro. METHODS We conducted a series of experiments using 17α-ethinylestradiol (EE) induced cholestatic rats and cultured HepG2 cells. Serum, bile, and liver samples were collected for biochemical and histological analyses. Bile acid composition in liver was analyzed by LC-MS/MS. The mechanisms underlying the hepatoprotective of baicalin were investigated by RT-PCR, Western blotting analyses and immunohistochemistry. RESULTS Baicalin showed obvious hepatoprotective effects in EIC rats by reducing serum bio?markers and increasing the bile flow rate, as well as by alleviating liver histology and restoring the abnormal composition of hepatic bile acids (BAs). In addition, baicalin protected against EE induced liver injury by up-regulation of the expres?sion of hepatic efflux transporters and down-regulation of hepatic uptake transporters. Furthermore, baicalin increased the expression of hepatic BA synthase (CYP27A1) and metabolic enzymes (Bal, Baat and Sult2a1) in EIC rats. We showed that baicalin significantly inhibited hepatic inflammatory responses in EIC rats through reducing elevated levels of TNF-α, IL-1β, IL-6 and NF-κB. Finally, we confirmed that baicalin maintains BA homeostasis and alleviates inflamma?tion through Sirt1/HNF-1α/FXR signaling pathway. CONCLUSION Baicalin protects against estrogen-induced cholestatic liver injury, and the underlying mechanism involved is related to activation of the Sirt1/HNF-1α/FXR signaling pathway.
ABSTRACT
Objective:To observe the expression of hepatocyte nuclear factor 1<italic>α</italic> （HNF1<italic>α</italic>）， proprotein convertase subtilisin/kexin type 9 （PCSK9） and low-density lipoprotein cholesterol （LDLR） in hypercholesterolemia rat liver， and investigate the mechanism of Shuangyu Tiaozhi Decoction regulating cholesterol metabolism and attenuating hypercholesterolemia. Method:After providing a high-fat diet for 4 weeks， 40 SD rats were selected， 8 of which were randomly selected as normal group and fed a normal diet， and the remaining 32 rats were fed a high-fat diet. The rats successfully established as hypercholesterolemic model， were randomized into 4 groups： model group， low dose of Shuangyu Tiaozhi decoction group （7.8 g·kg<sup>-1</sup>）， high dose of Shuangyu Tiaozhi decoction group （15.6 g·kg<sup>-1</sup>）， and simvastatin group （4 mg·kg<sup>-1</sup>）， with 8 rats in each group. The drugs were continuously given for 8 weeks. Serum total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C） were measured. The pathomorphological changes in liver were observed by hematoxylin and eosin （HE） staining. The immunohistochemistry was used to detect PCSK9 and LDLR expression in liver. The mRNA and protein expression levels of HNF1<italic>α</italic>， PCSK9 and LDLR were determined by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Result:Compared with normal group， the TC， TG， LDL-C levels in model group were significantly increased （<italic>P</italic><0.01）， the morphology showed obvious liver steatosis. The mRNA and protein expression of HNF1<italic>α</italic> and PCSK9 were increased （<italic>P</italic><0.05）， the mRNA and protein expression of LDLR was decreased （<italic>P</italic><0.05）. Compared with model group， the serum TC， TG， LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction high-dose group （<italic>P</italic><0.01）， the serum TC， LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction low-dose group and simvastatin group （<italic>P</italic><0.05，<italic>P</italic><0.01）， while no significant effect was observed on the serum HDL-C levels in each treatment group. The liver steatosis decreased in each treatment group. The mRNA and protein expression of HNF1<italic>α</italic> was obviously decreased in each treatment group （<italic>P</italic><0.05，<italic>P</italic><0.01）， the mRNA and protein expression of PCSK9 was obviously decreased in Shuangyu Tiaozhi decoction low and high-dose groups （<italic>P</italic><0.05，<italic>P</italic><0.01）， the mRNA expression of PCSK9 was significantly increased in the simvastatin group （<italic>P</italic><0.01）， while the protein expression showed a downward trend. The LDLR mRNA levels were significantly increased in each treatment group （<italic>P</italic><0.01）， the LDLR protein expression was significantly increased in Shuangyu Tiaozhi high-dose group （<italic>P</italic><0.01）， and showed an upward trend in Shuangyu Tiaozhi low-dose group and simvastatin group. Results of immunohistochemistry showed PCSK9 expression was weakly positive， the expression of LDLR was strongly positive in each treatment group. The therapeutic effect of Shuangyu Tiaozhi decoction high-dose group was more remarkable than simvastatin group， while there was no obvious difference between the Shuangyu Tiaozhi decoction low-dose group and simvastatin group. Conclusion:Shuangyu Tiaozhi decoction may reduce the blood lipid levels through HNF1<italic>α</italic>/PCSK9/LDLR signaling pathway， play an active role on regulating cholesterol metabolism and alleviating high-fat diet-induced hypercholesterolemia.
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Objective To investigate the expression of hepatocyte nuclear factor-1α (HNF-1α) and hepatocyte nuclear factor-4α (HNF-4α) in human hepatocellular carcinoma (HCC) and explore the function of HNF-1α and HNF-4α during HCC carcinogenesis and development. Methods Twenty-six specimens of hepatocellular carcinoma were collected. The expressions of HNF-1α and HNF-4α in HCC tissues and adjacent non-cancerous tissues were detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry staining. Results The mRNA levels of HNF-1α and HNF-4α were significantly lower in HCC tissues than that in adjacent non-cancerous tissues (0.818±0.371 vs. 0.383±0.102 for HNF-1α, P＜0.05;0.846±0.384 vs. 0.397±0.105 for HNF-4α, P＜0.05).The positive rates of HNF-1α and HNF-4α protein were significantly lower in HCC tissues than in adjacent non-cancerous tissues (92.3% vs. 42.3% for HNF-1α, P＜0.05;96.2% vs. 50.0% for HNF-4α, P＜0.05). The mRNA and protein expressions of HNF-1α and HNF-4α were correlated with tumor differentiation (P＜0.05). There was a negative correlation between HNF-1α and HNF-4α mRNA expressions in HCC tissues.Conclusion The expressions of HNF-1α and HNF-4α are down-regulated in HCC, which might be related to carcinogenesis and development of HCC.
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Objective: The aim of this study is to generate a mutation causing maturity-onset diabetes of the young (MODY) in Thai patients by insertion of a fourteen base-pair (bp) into a HNF-1a gene using a modified site-directed ligase-independent mutagenesis (SLIM) method. Methods: Two pairs of long- and short-tailed primers were designed to amplify a plasmid construct containing a HNF-1a and to insert a 14-bp at a desired position. Long-tailed primers contained the overhanging 14-nucleotide (nt) insert at their termini which were complementary to each other. Polymerase chain reactions (PCR) were performed in two separated tubes using different pairs of primers. After amplifications, PCR products from both tubes were pooled together, denatured and then re-annealed to allow formation of double stranded DNA molecules containing the 14-bp insert within HNF-1a. The pooled and reannealed PCR products without ligation were transformed into competent E.coli cells to generate a ligated recombinant plasmid with a 14-bp insertion. Results: Five of 14 bacterial colonies contained the desired recombinant plasmid with a 14-bp insertion within HNF-1a The efficiency of the method for generation of recombinant plasmid was about 36 percent. Conclusion: This method is simple and rapid to insert a long stretch of nucleotides into a plasmid construct containing a gene of interest at a desired position. A recombinant plasmid containing an insertion mutation in a HNF-1a gene was successfully generated, allowing an opportunity to perform functional study of the mutated gene.