| Liver fibrosis, an abnormal wound healing process in liver injury, whichis characterized by excessive scar formation due to over-accumulation ofcollagen. This process usually chronically occurs and finally leads to organdysfunction and death. As the key cellular source of over-accumulatedextracellular matrix, activation of HSCs has been identified as one of theessential dirven forces in liver fibrosis. This finding is a cornerstone toinvestigate the mechanism of fibrosis and develope novel therapeuticstrategies. Following acute or chronic liver tissue damage, HSCs transdiffer-entiate from quiescent, lipid droplet-containing cells toward myofibroblastlikecells, which are characterized by a decreased number of lipid droplets,increased proliferation and enhanced synthesis of extracellular matrixcomponents. Autophagy is a metabolic process that degrades and recyclesintracellular organelles and proteins with many connections to physiologicaland pathological processes. Recently, several studies were able to demonstratethat HSC activation is followed by an increased autophagic flux and that itsinhibition can partially inhibit the HSC myofibroblastic transition. Theseresults point to autophagy as a possible target in the attenuation of fibrogensisthrough alleviating HSC activation.C-Jun-N-terminal kinase (JNK) is a mitogen-activated protein kinasefamily (MAPK) member that is activated by diverse stimuli, includingcytokines (such as platelet-derived growth factor, tumor necrosis factor andinterleukin-1), reactive oxygen species (ROS), pathogens, toxins, drugs,endoplasmic reticulum stress, free fatty acids, and metabolic changes. JNK isactivated via3-tiered signaling modules comprising MAP kinase kinasekinases (MAP3Ks or MKKKs), MAP kinase kinases (MAP2Ks or MKKs), and MAP kinases (MAPKs, in this case the JNK). PDGF is the most potentproliferative cytokine for the HSCs. PDGF has been shown to transmit severalsignals in HSCs, including the JNK. At least50proteins have been identifiedas JNK substrates. Among these substrates, c-Jun is a representative target ofJNK. Upon activation, JNK induces multiple biologic events through thetranscription factor activator protein-1（AP-1） and transcription-independentcontrol of effector molecules.At present. the role of JNK signaling pathway in the autophagic processduring activation of hepatic stellate cells is unclear. Further study is necessaryto fill the knowledge gap.Objective: To investigate the effects of JNK inhibitor SP600125to thePDGF-mediated autophagy and activation of HSCs.Methods: Cells were grouped as follows:①Control group;②PDGFgroup;③SP600125group;④SP600125+PDGF group. Western blot wasused to determine the expressions of p-JNK, JNK, p-c-Jun and c-Jun. Acridineorange (AO) staining were used to abserve the autophagy activation of HSCs.Western blot and real-time PCR were used to detect the content of Beclin-1and Atg-5. LC3BⅡ and LC3BⅠexpressions were detected by western blot.α-SMA expressions were detected by immunocytochemistry and western blot.Results:1The phosphorylation levels of JNK and c-Jun PDGF-induced wereinihibited by SP600125in HSCs. Western blot was used to detect the proteinexpression ratios of p-JNK to JNK and p-c-Jun to c-Jun. The results showedthat the phosphorylation levels of JNK and c-Jun were significantly increasedin PDGF group than Control group (0.77±0.05vs0.34±0.02,0.92±0.52vs0.51±0.05)(p<0.01). SP600125+PDGF group and SP600125group comparedwith PDGF group and Control goup, the JNK (0.39±0.04vs0.77±0.05,0.24±0.02vs0.34±0.02) and c-Jun phosphorylation levels (0.21±0.04vs0.92±0.52,0.14±0.01vs0.51±0.05) were significantly lower respectively 2The PDGF-induced autophagy of HSCs were inihibited by SP600125.In acridine orange staining, the pre area of red fluorescence were counted. Theresults showed that PDGF group than Control group was significantly higher(130.00±24.10vs54.60±8.30)(p<0.05). SP600125+PDGF group andSP600125group compared with PDGF group and Control goup, the pre areaof red fluorescence were significantly lower respectively (50.20±6.60vs130.00±24.10,29.40±6.60vs54.60±8.30)(p<0.05); Western blot was usedto detect the protein expression ratios of LC3BⅡ to LC3BⅠ. The resultsshowed that the relative expressions of LC3-Ⅱ/LC3-Ⅰ were significantlyhigher in PDGF group than Control group (1.29±0.21vs0.66±0.05)(p<0.05).SP600125+PDGF group and SP600125group compared with PDGF groupand Control goup, the relative expressions of LC3-Ⅱ/LC3-Ⅰ weresignificantly lower respectively (0.35±0.03vs1.29±0.21,0.33±0.05vs0.66±0.05)(p<0.05); Western blot was used to detect the protein expression ofBeclin-1and Atg-5. The results showed that the expressions of Beclin-1andAtg-5were significantly increased in PDGF group than Control group(746.10±46.33vs310.20±31.80,1778.00±130.00vs562.90±60.90)(p<0.01).SP600125+PDGF group compared with PDGF group, the expressions ofBeclin-1and Atg-5were significantly lower (197.50±26.99vs746.10±46.33,714.20±47.70vs1778.00±130.00)(p<0.01). Real-time PCR was used todetect the mRNA expression of Beclin-1and Atg-5, and the results wereconsistent with western blot.3The expressions of α-SMA were inihibited by SP600125in HSCs.α-SMA expressions were detected by immunocytochemistry and western blot.The results showed that the expressions of α-SMA were significantlyincreased in PDGF group than Control group (1566.00±116.50vs757.70±72.87)(p<0.01). SP600125+PDGF group compared with PDGF group, theexpressions of α-SMA were significantly lower (582.10±60.48vs1566.00±116.50)(p<0.01).Conclusion: Specifically blocking JNK pathway curbs the PDGF-induced autophagy and activation of hepatic stellate cells. |
PDF Full Text Request