The Effects Of Liposaccharide And CTGF ASON On CTGF MRNA Expression In Hepatic Stellate Cells In Vitro

Hepatic fibrosis is a fundamental pathological process in chronic liver diseases, but the pathogenesis of hepatic fibrosis remains to be established. There are many factors regulating hepatic fibrosis, among which endotoxemia is one of the important triggering factors. Hepatic stellate cell(HSC) is the key cell element, capable of secreting many cytokines implicated in hepatic fibrosis. Connective tissue growth factor (CTGF) can promote fibroblast proliferation, differentiation and extracellular matrix formation. Abnormal expression of CTGF plays a major role in fibrosis of many organs. This research studied the interrelationship between CTGF expression level and the degree of hepatic fibrosis in activated HSC in vitro, and explored the possibility of CTGF antisense olgionucleotide (ASON) as a novel anti-fibrosis therapeutical modality.Objects: 1. To study effects of lipopolysaccharide (LPS) on CTGF mRNA expression, and to explore its association with hepatic fibrosis; 2. To study the effects of CTGF ASONs coated by cation lipofectin on HSC CTGF mRNA and fibronectin (FN) mRNA expression.Methods: 1. In vitro culture and passage of HSC. Activated HSC was confirmed by immunostaining against desmin and smooth muscle a-actin (a-SMA). 2. HSCs were first treated by LPS of different concentrations and for different periods of time. FN protein expression was evaluated by immunohistochemistry and CTGF mRNA expression levels in different time points were studied by semiquatitative RT-PCR; 3. The differences of CTGFand FN mRNA expression levels between CTGF ASONs treated HSC and HSC treated with both CTGF ASONs and LPS were assessed by semiquatitative RT-PCR.Results: The percent of activated HSCs with positive desmin and a-SMA staining was over 90% after in vitro culture and passage. 2. FN was stained dark brownish in the cytoplasma of HSC treated by LPS (10ug/ml) for 24 hours, which was much more remarkable than that in control cells. 3. LPS could upregulate the expression of CTGF mRNA and there seemed to exist a dose-effect relationship. The CTGF mRNA expression level was increased by one fold with LPS treatment at the concentration of 1 jig/ml as compared with control HSC with no LPS stimulation. In addition, when stimulated with fixed LPS concentration, the expression of CTGF mRNA increased with increasing LPS stimulation time, became evident after 6-hour LPS treatment and reaching its peak level at 24-48 hours. 4. The expression levels of CTGF mRNA and FN mRNA were downregulated in CTGF ASONs treated HSCs compared to that in control cells. There were no alteration of CTGF and FN mRNA expression levels in HSCs first treated by CTGF ASONs for 18 hours and then LPS treatment for 24 hours.Conclusions: 1. The fact that LPS can up-regulate both the expression of FN and CTGF in HSCs suggests that CTGF might play an important role in the pathological process of LPS-induced live fibrosis. 2. CTGF ASONs can inhibite CTGF and FN mRNA expression, therefore CTGF itself may turn out to be a new target for therapeutic intervention in liver fibrosis.