Effect of protein kinase C/transforming growth factor beta 1 pathway on activation of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of protein kinase C (PKC)/transforming growth factor beta 1 (TGF beta1) pathway on activation of hepatic stellate cells (HSC).</p><p><b>METHODS</b>HSC rHSC-99 cell line was used in three groups in this study. Group A served as a control. In group B the HSC were incubated with PKC agonist PMA (0.5 micromol/L), and in group C the cells were incubated with PKC inhibitor calphostin C (100 nmol/L). The PKC activities were detected at different incubation time points (0, 3, 6, 12 and 24 h). Western blot and RT-PCR were used to detect the expression of TGF beta1, Smad 4, collagen type I, III and alpha-smooth muscle actin (alpha-SMA) at the 24 h point. Cell proliferation was assessed by MTT colorimetric assay.</p><p><b>RESULTS</b>PMA increased the activity of PKC significantly, whereas calphostin C inhibited the activity of PKC. The increased activity of PKC promoted the HSC to express TGF beta1, Smad 4, collagen type I, III and alpha-SMA. In comparison with the controls, the expressions of TGF beta1, Smad 4, collagen type I, III and alpha-SMA increased 4.8, 13.1, 2.4, 1.8 and 1.3 fold respectively (P < 0.01). PKC promoted the proliferation of HSC. The above effects were inhibited by the inhibition of PKC activity.</p><p><b>CONCLUSION</b>Changing of PKC activity can regulate and control the expression of TGF beta1, which may play a role in regulating the activation of HSC.</p>

Downregulation of transforming growth factor-beta1 and platelet-derived growth factor gene expression by interleukin-10 in murine hepatic stellate cells in response to experimental liver injury / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of interleukin-10 (IL-10) on the expression of transforming growth factor-beta(1) (TGFbeta(1)) and platelet-derived growth factor (PDGF) in hepatic stellate cells (HSC) during liver injury.</p><p><b>METHODS</b>The adenovirus vector (the titer was 1 x 10(7) efu/ml) encoded IL-10 gene was used to transfect the rat via the vein of caudal. At the same time, CCl(4) was injected into rat by a hypodermic injection. These processes went on twice a week. After eight weeks, the liver were perfused with collagenase IV and purified by density gradient centrifugation with Nycodenz for separate HSC. The level of IL-10 was measured by ELISA method; The expression of PDGF and TGFbeta(1) in HSC was detected by semi-quantitative RT-PCR and Western-blot methods.</p><p><b>RESULTS</b>The level of IL-10 in therapy group (adenovirus vector encoding IL-10 gene group) was higher than that in non-therapy group (adenovirus vector without IL-10 gene and PBS group); The expression of TGFbeta(1) mRNA, TGFbeta(1) protein and PDGF mRNA, PDGF protein in therapy group were significantly lower than that in non-therapy group (P < 0.05).</p><p><b>CONCLUSION</b>Downregulating the TGFbeta(1) and PDGF expression could be the passageway by which IL-10 alleviate the degree of proliferation and activation in hepatic stellate cells.</p>

An in vivo study on interlukin-10 inhibiting the expression and secretion of collagen I and IV in hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate the effect of interlukin-10 (IL-10) on expression and secretion of collagen I, IV in rat's hepatic stellate cells (HSC) of livers injured by CCl4.</p><p><b>METHOD</b>The adenovirus vector encoded IL-10 gene was used to transfect rats with liver injury via the caudal veins. HSC were isolated and purified from the rat livers by collagenase IV perfusion and density gradient centrifugation with Nycodenz. The expression of collagen I, IV mRNA in HSC was detected by semi-quantitative RT-PCR method and the secretion of collagen I, IV in culture serum of HSC by ELISA method. The quantity of collagen was measured in the van Gieson stained histological liver preparations.</p><p><b>RESULTS</b>The expression and secretion of collagen I, IV in the adenovirus vector encoding IL-10 gene group were significantly lower than those in the adenovirus vector without IL-10 gene group and the control group (P < 0.05). The quantity of collagen in the treatment group was lower than that in the control group.</p><p><b>CONCLUSION</b>IL-10 can inhibit collagen I, IV expression and secretion in rat HSC.</p>

Inhibitory effect of retroviral vector containing anti-sense Smad4 gene on Ito cell line, LI90 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transforming growth factor-beta1 (TGF-beta1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.</p><p><b>METHODS</b>The recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.</p><p><b>RESULTS</b>mRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P < 0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.</p><p><b>CONCLUSIONS</b>The antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-beta1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix (ECM). Our results may provide a basis for the development of antifibrotic gene therapy.</p>

The effect of somatostatin analogue on expression of connective tissue growth factor gene of rat hepatic stellate cells / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of somatostatin analogue-octreotide (OCT) on expression of connective tissue growth factor (CTGF) gene of murine hepatic stellate cells (HSCs) in vitro.</p><p><b>METHODS</b>HSCs separated from Sprague Dawley rats by in situ perfusion and Nycodenz gradient were divided into 5 groups. HSCs in 4 out of 5 groups were co-cultured with octreotide at different dosages, and the remaining group served as control. The expression of CTGF and TGF-beta mRNA were assessed by reverse transcription-polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>OCT down-regulates the expression of CTGF and TGF-beta mRNA in HSCs. The effect is increased with a dose dependent manner.</p><p><b>CONCLUSIONS</b>OCT could exert the inhibitory effect on HSCs by down-regulating the expression of CTGF and TGF-beta. This provides a potential for the prevention and management of hepatic fibrosis.</p>

Expression of 5-hydroxytamine receptors in hepatic stellate cell and action of 5-hydroxytamine on biological characteristics of hepatic stellate cell / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of 5-hydroxytamine receptors in hepatic stellate cells HSCs and action of 5-hydroxytamine on biological characteristics of HSC.</p><p><b>METHODS</b>Liver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate hepatic stellate cell. RT-PCR was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. HSCs were cultured on silicone membrane. The effect of 5-hydroxytamine, ketanserin and ondanosetron on cell contraction were studied.</p><p><b>RESULTS</b>HSC expressed 5-hydroxytamine receptors subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P < 0.05). This was antagonized by ketanserin, not by ondanosetron. 5-hydroxytamine induced cell contraction in a dose-dependant manner. Ketanserin antagonized this action, but ondanosetron did not.</p><p><b>CONCLUSIONS</b>HSCs express 5-hydroxytamine receptors. 5-hydroxytamine could affect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.</p>