| Liver fibrosis, a wound-healing response to a variety of chronic stimuli,is characterized by excessive deposition of extracellular matrix （ECM）proteins, of which type I collagen predominates. This alters the structure of theliver leading to organ dysfunction. Liver fibrosis is easily reversible, whileliver sclerosis is hardly reversible. Thus liver fibrosis is taken as the treatmentfocus. Nowadays, lack of effective treatment makes it more important toinvestigate the pathogenesis of liver fibrosis.Hepatic stellate cells （HSCs） are primarily responsible for excess ECMdeposition during liver fibrosis. Two important aspects are involved inmediating the fibrogenic response: the HSCs become directly fibrogenic bysynthesizing ECM; the activated HSCs proliferate, effectively amplifying thefibrogenic response. In the quiescent state, HSCs are characterized bysignificant expression of desmin and vitamin A storage. Following liver injury,HSCs lose their vitamin A content; increase the expression of α-smoothmuscle actin （α-SMA）, acquire a myofibroblast-like phenotype losing theirtypical star-shape, become proliferative, motile, pro-fibrogenic, contractile andshow abundant rough endoplasmic reticulum. Recently, it was found thatautophagy was related to lipid droplet degradation, which provided energyduring HSCs activation. In contrast, inhibiting autophagy was able to restrainthe activation of HSCs accompanied with the expressions of α-SMA andcollagen Ⅰ reduced.Autophagy is derived from the Greek roots “auto”（self） and “phagy”（eating） and broadly refers to the cellular catabolic processes in whichcytoplasmic target material is transported to lysosomes for degradation. Thelysosome is the primary organelle for degradation through its wide array ofresident acid hydrolases. Classical autophagy process can be largely divided into three major steps that require specific autophagy-related （Atg） genes,which include initiation of autophagosome formation, autophagosomemembrane elongation and completion and autophagosome maturation throughfusion with lysosomes and cargo degradation through lysosomal enzymes andrecycling. Chloroquine （CQ） inhibits lysosomal acidification and thereforeprevents autophagy by blocking autophagosome fusion and degradation.At present the study between liver fibrosis and autophagy mainly focuson the fact that autophagy promotes HSCs activation through providing energy.However, the effect of autophagy to activated HSCs is unclear. The presentstudy aims to clarify the influence of autophagy to the expressions of collagenⅠ, collagen Ⅲ, MMP-2, TIMP-2, MMP-13and TIMP-1in activated HSCs.Objective: To investigate the effects of CQ on collagen Ⅰ, collagen Ⅲ,MMP-2, TIMP-2, MMP-13, TIMP-1changes on activated HSCs.Methods: Activated HSCs-T6with TGF-β1and then treated with threedosages of CQ for24h. Cells were grouped as follows:①Control group,cells were cultured with DMEM contained10%FBS, and added the samevolume of TGF-β1solvent when it’s time to activate HSCs.②TGF-β1group,added TGF-β1with the concentration of20ng/ml, when the cells come to70%confluence.③TGF-β1+CQ15μmol/L group, added CQ with theconcentration of15μmol/L to HSCs after activation.④TGF-β1+CQ30μmol/L group, added CQ with the concentration of30μmol/L to HSCs afteractivation.⑤TGF-β1+CQ60μmol/L group, added CQ with theconcentration of60μmol/L to HSCs after activation.MTT was used to detect cell viability. Western blot was used todetermine the expressions of LC3-Ⅱ/LC3-Ⅰ a nd P62.Collagen Ⅰandcollagen Ⅲ expressions weredetected by immunocytochemistry, western blotand real-time Q-PCR. Western blot and real-time Q-PCR were used to detectthe contents of MMP-2, TIMP-2, MMP-13and TIMP-1.Results:①The intervening concentration of TGF-β1was chosenaccording to the cell viability. The cell viability was strengthened with theincrease of TGF-β1concentration. However, it was increased slowly after the concentration of TGF-β1come to20ng/ml which was chosen in the presentstudy.②Western blot was used to detect the relative expressions of LC3-Ⅱ/LC3-Ⅰa nd P62proteins. The results showed that the expressions of LC3-Ⅱ/LC3-Ⅰ in TGF-β1+CQ groups were markedly higher than that in TGF-β1group （1±0.1,1.5±0.3,2.4±0.17vs0.61±0.12, P<0.01）. There were significantdifferences among the TGF-β1+CQ groups （P<0.01）. The expressions of P62protein in TGF-β1+CQ groups were significantly higher than in TGF-β1group（5.9±0.25,6.2±0.31,6.1±0.43vs4.1±0.33, P<0.01）. No marked differencewas found among TGF-β1+CQ groups （P>0.05）.③Cell viabilities in theTGF-β1+CQ groups were markedly lower than in TGF-β1group （89%,48%,27%vs115%, P<0.01）. And there were obviously differences among theTGF-β1+CQ groups （P<0.01）.④The α-SMA expressions in TGF-β1group（1.23±0.21） and TGF-β1+CQ groups （1.18±0.16,1.2±0.23,1.31±0.33） weresignificantly higher than the control group （0.84±0.12）, P<0.05. But nostatistical difference was found between TGF-β1group and TGF-β1+CQgroups, P>0.05.⑤The results of collagen Ⅰ expression detected byimmunocytochemistry showed that the positive area densities were obviouslyincreased in the TGF-β1+CQ groups compared to the TGF-β1group. And thesimilar tendency was also found in collagen Ⅲ expression.⑥Western blotwas used to determine the expressions of collagen Ⅰ and collagen Ⅲ. Theresults showed that the expressions of collagen Ⅰ in TGF-β1+CQ groups（1.43±0.17,1.6±0.14,1.95±0.2） were markedly higher than the TGF-β1group（1.12±0.11）, P<0.01. The TGF-β1+CQ30μmol/L group was significantlyhigher than the TGF-β1+CQ15μmol/L group, P<0.01. The TGF-β1+CQ60μmol/L group was significantly higher than the TGF-β1+CQ30μmol/L group,P<0.05. The collagen Ⅲ expressions inTGF-β1+CQ groups （1.84±0.13,3.1±0.23,3.9±0.32） were obviously higher than the TGF-β1group （1.28±0.2）,P<0.01. And there were also statistical differences among the TGF-β1+CQgroups, P<0.01.⑦Real-time Q-PCR was used to assay relative mRNAexpression levels of collagen Ⅰ and collagen Ⅲ using the method of foldincrease (2-△△Ctmethod). The expression value of control group was arbitrarily assigned an expression value of1. The expressions of collagen Ⅰ in theTGF-β1+CQ groups （2.1±0.11,2.5±0.08,2.8±0.13） were markedly higher thanthe TGF-β1group （1.8±0.06）, P<0.05. There were also statistical differencesamong TGF-β1+CQ groups, P<0.05. The expressions of collagen Ⅲ in theTGF-β1+CQ groups （2.7±0.07,3±0.17,3.7±0.09） were markedly higher thanthe TGF-β1group （2.1±0.13）, P<0.05. There were also statistical differencesamong TGF-β1+CQ groups, P<0.05.⑧Western blot showed that the relativeexpressions of MMP-2in TGF-β1+CQ groups （0.69±0.06,0.71±0.1,0.74±0.09） and TGF-β1group （0.67±0.08） were markedly lower than thecontrol group （1.01±0.11）, P<0.05. But no statistical differences were foundbetween the TGF-β1+CQ groups and TGF-β1group, P>0.05. And there wereno markedly differences among the three TGF-β1+CQ groups, P>0.05. Theexpressions of TIMP-2in TGF-β1+CQ groups （1.31±0.11,1.58±0.17,1.82±0.16） were significantly higher than the TGF-β1group （1.19±0.13）,P<0.05. There were also significant differences among the three TGF-β1+CQgroups, P<0.05.⑨Real-time Q-PCR was used to detect the relative mRNAlevels of MMP-2and TIMP-2. The relative mRNA levels of MMP-2inTGF-β1+CQ groups （2.8±0.1,3.2±0.14,3.8±0.17） were markedly higher thanthe TGF-β1group （2.61±0.11）, P<0.05. There were also statistical differencesamong the three TGF-β1+CQ groups, P<0.05. The similar tendency was alsofound in the relative mRNA levels of TIMP-2.⑩Western blot showed thatthe relative expressions of MMP-13in TGF-β1+CQ groups （0.84±0.1,0.55±0.07,0.46±0.09） were markedly lower than the TGF-β1group（1.27±0.07）, P<0.05. There were also markedly differences among the threeTGF-β1+CQ groups, P<0.05. The expressions of TIMP-1in TGF-β1+CQgroups （1.25±0.04,1.48±0.12,1.6±0.05） were significantly higher than theTGF-β1group （1.02±0.09）, P<0.01. There were also significant differencesamong the three TGF-β1+CQ groups, P<0.05.○11Real-time Q-PCR was usedto detect the relative mRNA levels of MMP-13and TIMP-1. The relativemRNA levels of MMP-13in TGF-β1+CQ groups （2.2±0.06,1.8±0.03,1.2±0.03） were markedly lower than the TGF-β1group （3.1±0.12）, P<0.05. There were also statistical differences among the three TGF-β1+CQ groups,P<0.05. The relative mRNA levels of TIMP-1in TGF-β1+CQ groups （3.3±0.1,3.8±0.04,4.8±0.07） were markedly higher than the TGF-β1group （2.7±0.05）,P<0.01. There were also statistical differences among the three TGF-β1+CQgroups, P<0.05.Conclusion: Inhibiting autophagy with CQ was able to increase theexpressions of collagen Ⅰ and collagen Ⅲ in dose-dependent fashion in HSCs,which was probably related to up-regulate the expressions of TIMP-1andTIMP-2, down-regulate MMP-13. |
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