The Role Of Autophagy In Hepatic Stellate Cell Fibrosis. The Characteristics Of Dyslipidemia In Primary Biliary Cholangitis

Background and objectiveLiver fibrosis is a pathological process caused by the synthesis and degradation of the extracellular matrix(ECM)and unbalanced sedimentation in the liver after sustained damage and repair reactions.Hepatic stellate cells(HSC)are the central part of liver fibrosis,and the activation of HSC can be converted into the myofibroblasts that synthesize and secrete large amounts of collagen fibers.Transforming growth factor-?1(TGF-?1)is the most critical cytokine that mediates liver injury and fibrosis.Autophagy is a lysosomal-mediated process of degradation of intracellular proteins and organelles,and is an important mechanism for the regulation of survival in cells.Many studies have shown that cell autophagy plays an important role in the development of liver fibrosis.Primary biliary cholangitis(PBC)is an autoimmune hepatic disease with unknown etiology.Its main feature is intrahepatic cholestasis,and the ultimate outcome is liver fibrosis and cirrhosis.Our team's early research found that TGF-?1 in the liver tissue of PBC may regulate the production of alpha-smooth muscle actin(SMA)through the autophagy level.Therefore,the specific mechanism of TGF-?1 and autophagy in the formation,development and progression of liver fibrosis is explored.It is of great significance to treat liver fibrosis with more effective and safe TGF-?1 or autophagy as targets.The research results will provide a scientific basis for delaying the progression of liver fibrosis or stopping the pathological process of liver disease.The purpose of this study was to investigate the relationship among autophagy,TGF-?1 and liver fibrosis.MethodsLX-2 was cultured and passaged in DMEM complete medium containing 10%FBS and disposed in different ways:(1)LX-2 was co-cultured with DMEM complete medium for 48 hours(h),and LX-2 was collected at 0,1,3,6,9,12,24,36,and 48 hours.(2)After adding TGF-?1 to a final concentration of 10 ng/ml for 48 hours,LX-2 was collected at 0,1,3,6,9,12,12,24,and 48 hours.(3)Add TGF-?1 to a final concentration of 10 ng/ml and co-culture with LX-2 for 48 h,add autophagy inhibitor BA1 at 20 h to a final concentration of 3 nmol/L,10 nmol/L,and CQ to a final concentration of 10 ?mol L/L,30 umol/L,3-MA to a final concentration of 300umol/L,1000umol/L respectively,collected LX-2 at 48h.(4)LX-2 was co-cultured with DMEM complete medium for 48h,at 20 h,BA1 was added to a final concentration of 10 nmol/L,and CQ to a final concentration of 30 umol/L respectively,collected LX-2 at 48h.(5)TGF-?1 was added to a final concentration of 10 ng/ml and co-cultured with LX-2 for 48 h.After 20 h,DMEM complete medium was changed to a balanced salt solution,LX-2 was starved to activate autophagy for 4 h,LX-2 cell protein samples were collected at this time.At the same time,balanced salt solution was replaced with complete medium and cultured for 48 hours after the addition of TGF-?1 and protein samples were collected for testing.(6)The protein expression of LC3B-II,Fibronectin and alpha-SMA in different treatment groups were detected by Western Blotting.(7)The relative expression levels of Fibronectin and alpha-SMA genes in each group were detected by RT-PCR.Results(1)LX-2 had low levels of autophagy in the basal state,and it became more obvious as the culture time prolonged,but there was no significant change in the expression of Fibronectin and alpha-SMA proteins.(2)TGF-?1 stimulated LX-2 autophagy to begin at 3h,peaked at 24h and decreased at 48h;the expression of Fibronectin and alpha-SMA proteins increased with time,and increased significantly at 48h.(3)After stimulated by TGF-?1 and autophagy,the expression of LC3B-? protein in BA1 and CQ groups with high concentration increased.After the addition of 3-MA,an early inhibitor of autophagy,the content of LC3B-II protein was significantly reduced,while the expression of Fibronectin and alpha-SMA proteins was increased due to the inhibition of autophagy.(4)Under normal culture conditions,the expression of LC3B-II protein increased after autophagy inhibition,and the expression of Fibronectin and alpha-SMA proteins increased with the inhibition of autophagy.(5)After TGF-?1 stimulation,autophagy induced by starvation increased the content of LC3B-? protein,and the expression of Fibronectin and alpha-SMA proteins decreased with the activation of autophagy.(6)The relative expression of Fibronectin and alpha-SMA genes in TGF-?1 stimulation group was significantly higher than that in basal state group,and the difference was statistically significant,(p<0.01);Compared with TGF-?1 group,the relative expression levels of Fibronectin and alpha-SMA genes in TGF-?1+BA1 group,TGF-?1+CQ group,TGF-?1+3-MA group,TGF-?1+starved group respectively had no significant difference.ConclusionLX-2 has low levels of autophagy in the basal state without significant fibrosis;TGF-?1 can activate LX-2 to autophagy and promote fibrosis;Inhibition of LX-2 autophagy under TGF-?1 stimulation can promote fibrosis and activation of autophagy inhibit fibrosis.ObjectiveTo investigate the clinical characteristics of dyslipidemia in patients with primary biliary cholangitis(PBC);To investigate the characteristics of dyslipidemia in primary biliary cholangitis(PBC),analyze its distribution and the difference among PBC.PBC complicated with Sjogren's syndrome and simple dyslipidemia patients,evaluate its clinical significance.MethodsThe clinical and laboratory data of 136 hospitalized and outpatient PBC patients in**Hospital from 2010 to 2016 were retrospectively analyzed:A total of 100 PBC complicated with dyslipidemia patients were enrolled.In addition,83 patients with simple dyslipidemia?PBC complicated with Sjogren's syndrome were enrolled as controls.The clinical features of PBC-associated dyslipidemia were analyzed,and the differences between these two groups were evaluated.Results(1)The distribution of dyslipidemia Among the 136 patients with PBC,100(74%)had abnormal serum lipids.The incidence of dyslipidemia was higher than the same age group.The distribution of dyslipidemia in PBC patients in terms of total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and triglyceride(TG)increased by 61%(59/96),58%(48/83)and 47%(46/97),respectively,while high-density lipoprotein cholesterol(HDL-C)decreased by 26%(21/82).For controls,in PBC complicated with Sjogren's syndrome group,the corresponding rate was 62%(18/29)?48%(13/27)?34%(10/29)and 19%(5/27),while in Simple dyslipidemia group,the corresponding rate was 61%(51/83),70%(58/83),37%(31/83)and 25%(21/83),respectively.(2)Clinical characteristics of PBC According to the clinical classification of dyslipidemia,high TC,TG,LDL-C and mixed hyperlipidemia accounted for 14%,19%,10%and 57%in PBC group,respectively.In mixed hyperlipidemia group,the level of alanine aminotransferase(ALT,62 vs.101,P=0.04),aspartate aminotransferase(AST,81 ±65 vs.59:± 24,P=0.04),and total bile acid(TBA,6.5 vs.13.6,P=0.04),and the positive ratio of AMA(82%vs.50%,P=0.01)in the mixed hyperlipidemia group were higher in the high TC group,and the difference was statistically significant.Serum Lipid with clinical and laboratory correlation analysis:TG and LDL-C levels were positively correlated with pruritus,while TG and TC were positively correlated with liver enzymes,bile enzymes and bile acids.(3)Blood lipid comparison between groups Compared with Simple dyslipidemia group,TG and HDL-C levels were higher,while LDL-C level was lower,the difference was statistically significant.The incidence of LDL-C in the patients with PBC combined with SS was lower than that in the simple hyperlipidemia group,and the difference was statistically significant(P=0.04).The mainly dyslipidemia of PBC was high TC,but was high LDL-C in the simple hyperlipidemia group.In terms of clinical classification and comparison,PBC combined with dyslipidemia group was more likely to present simple TC increase(14%vs 5%,P=0.046)and simple TG increase(19%vs 8%,P=0.042)than the simple dyslipidemia group,but with the lower proportion of mixed hyperlipidemia(57%vs 77%,P=0.004),the differences were statistically significant.The level of TG(2.36±2.13 vs 1.57±0.75,P=0.001)and HDL-C(1.64±0.75 vs 1.31 ±0.38,P=0.001)in PBC with dyslipidemia group was higher than that of the simple dyslipidemia group,but the level of LDL-C(3.27±1.22 vs 3.61 ±0.69,P=0.033)was lower,the difference was statistically significant.(4)Clinical comparison between groups Compared with the PBC patients with normal serum lipids,the incidence of pruritus(26%vs 8%,P=0.032).serum bilirubin(TBIL17.3 vs 14.5,P=0.02;DBIL 5.5 vs 4.4,P<0.04)and ALP/GGT level(GGT 193 vs 105.5,P=0.04;ALP 183 vs 135,P=0.048)were higher in the dyslipidemia group,and the difference was statistically significant(P<0.05).Compared with PBC combined with SS group,the incidence of dry mouth and dry eye is low,the incidence of hyperbilirubinemia is high,the positive rate of anti mitochondrial antibody is low,the level of platelets and albumin is high,and the difference is statistically significant between the simple PBC group and the combined group.Conclusions(1)The proportion of dyslipidemia in PBC patients was higher than that in normal population.Increased TC and LDL-C were more common in PBC.The main PBC dyslipidemia is mixed hyperlipidemia.The patients of PBC with mixed hyperlipidemia is more severe than other types of hyperlipidemia.The severity of dyslipidemia is more related to the degree of liver dysfunction.(2)The distribution of dyslipidemia in PBC was different from that of the simple dyslipidemia.The difference of clinical classification and blood lipid level between the two groups was obvious.PBC with dyslipidemia were prone to present with high TG blood than simple dyslipidemia,as well as with the higher level of TG and HDL-C,lower level of LDL-C.The incidence of LDL-C in PBC combined with SS patients is lower than that in simple hyperlipidemia group.There was no significant difference in the distribution of dyslipidemia and the level of lipid between the patients with simple PBC and the combined SS.Patients with SS were susceptible to dry eye and dry mouth,positive anti mitochondrial antibody,but low incidence of hyperbilirubinemia,whatsmore the level of platelets and albumin was lower than that of the simple PBC group.