Effect of Kupffer cell blockade on activation of mitogen-activated protein kinases in response to lipopolysaccharide

AIM: Using the mouse model of lipopolysaccharide(LPS) attack,we study the effect of Kupffer cell (KC) blockade on the activation of mitogen-activated protein kinases(MAPKs) signal transduction pathway induced by LPS.METHODS: GdCl₃ (10 mg/kg) or the same volume of NS was continually injected intravenously at 48 h and 24 h before LPS (5 mg/kg) was injected into the male mice of Kunming species.The liver was then took out and KCs were isolated 30 minute after LPS was injected.The KCs isolated from the mice were cultured,and pretreated with GdCl₃ (100 μmol/L) for 1 h.The culture medium containing LPS (100 μg/L) was added and continuously incubated for 30 minute.The protein expression and phosphorylation level of ERK1/2 and p38MAPK in liver or KCs were assayed in vivo and in vitro,and effect of GdCl₃ on the phagocytosis function was observed,respectively.RESULTS: LPS induced the protein phosphorylation of ERK1/2 and p38MAPK in KCs or liver,no effect on the protein expression was observed.GdCl₃ treatment inhibited LPS-induced KCs activation and secretion of TNF-α,however,it had no effect on ERK1/2 and p38MAPK in KCs or liver,neither at the protein expression nor the phosphorylation.KCs secreted a few TNF-α with short time treatment with GdCl₃ alone in vitro.CONCLUSION: KC blockade with GdCl₃ alleviates LPS-induced KCs activation and the release of TNF-α not through modulating intracellular ERK1/2 or p38MAPK signal transduction pathways.We presume that GdCl₃ might reduce liver injury through cross talk of other intracellular signal transduction pathways (JNK,NF-кB,GPCR,etc).     

Effect of LPS on phagocytosis of rat Kupffer cells in vitro

AIM:To investigate the effect of LPS on phagocytosis of Kupffer cells in vitro. METHODS:Isolated Kupffer cells were treated with LPS in vitro. The phagocytosis, microfilament, microtubules and apoptosis of Kupffer cells were determined by the beads phagocytosis test, fluorescence staining, fluorometry and flow cytometric analysis.RESULTS:LPS enhances the phagocytosis, actin content, microtubules fluorescence density of Kupffer cells in vitro, while at a large dose or for a long time, it lessened the phagocytosis increasing or phagocytosis, inhibites the microfilament and microtubules expression, and induced apoptosis.CONCLUSION:LPS enhances the phagocytosis of Kupffer cells in vitro, but in large amount, it inhibites the phagocytosis of Kupffer cells, which is probably related to LPS -induced microfilament, microtubules expression changes and apoptosis in Kupffer cells.     

The expression of CD14 in rat Kupffer cells

AIM: To explore the expression of CD14 in rat Kupffer cells (KCs). METHODS: In rat KCs induced by LPS or the mediators from KCs induced by LPS, the changes of CD14 expression were measured by RT-PCR and immunohistochemistry.The expressions of TNFα mRNA、IL-6 mRNA or the concentrations of TNFα、IL-6 were estimated by in situ hybridization and radioimmunoassay, respectively. RESULTS: LPS increased the expression of CD14 in KCs in a dose-dependent fashion (LPS, 1 μg/L-10 mg/L) and in a time-dependent fashion(0.5 h-24 h, peaked at 3-6 hours). While the expression of CD14 in KCs stimulated by the active mediators from KCs which had been exposed to LPS 1 hour were obviously increased. CONCLUSIONS: There was a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14. It is implied that the increase in CD14 expression may be induced by LPS and the cytokines produced by KCs, it also reveals that there is a auto-regulated loop in CD14 expression.     

Lipopolysaccharide stimulates TNF-α and endothelin-1secretion from cultured rat Kupffer cells

AIM: To investigate lipopolysaccharide (LPS) stimulated cytokine secretion from normal rat Kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured. Tumor necrosis factor -α (TNF-α) and endothelin-1 (ET-1) secreted by LPS stimulated Kupffer cells were detected. RESULTS: LPS had an stimulative effect on Kupffer cell activity. LPS in definite concentrations promoted Kupffer cell secretion. CONCLUSION: LPS promotes Kupffer cell secretion, which may be associated with liver injury induced by LPS.     

Changes of Kupffer cells during tree shrew chronically infected with hepatitis B virus

AIM：To explore the changes and significance of Kupffer cells in the process of tree shrew chronically infected with hepatitis B virus (HBV). METHODS：The animals were divided into 3 groups. Group A consists of 6 tree shrews that were identified as persistently infected with HBV; group B consists of 3 tree shrews that were suspected as persistently infected with HBV; group C consists of 4 tree shrews that were not inoculated with HBV and were applied as normal controls. Liver biopsies were collected regularly from all animals, and the Kupffer cells were isolated, purified and primarily cultured. The techniques of flow cytometry, immunohistochemistry, lysosomal fluorescent probe staining and real-time RT-PCR were applied to determine the number and function of these Kupffer cells. RESULTS：The result showed that the count and proportion of CD163+ cells in group A were significantly higher than those in group B and group C (P<0.05). Meanwhile, the fluorescence intensity levels of lysosomal, the number of lysozyme-positive cells and the mRNA expression level of TNF-α in the Kupffer cells in group A were significantly lower than those in group B and group C (P<0.05). CONCLUSION：Kupffer cells may play a regulatory role during host’s chronic HBV infection.     

Effect of miR-133 targeting NLRP3 on inflammatory activation of mouse Kupffer cells

AIMTo explore the effect of microRNA-133 (miR-133) targeting nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) on the inflammatory activation of Kupffer cells (KCs). METHODSThe KCs were isolated from mouse liver and identified. After successful identification, 1 mg/L lipopolysaccharide (LPS) was used to induce the KCs transfected with miR-133 inhibitor or miR-133 mimic. The mRNA expression levels of miR-133 and NLRP3 were detected by RT-qPCR. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cell culture medium were measured by ELISA. The protein levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1 were determined by Western blot. TargetScan was used to find the binding site of miR-133 and 3'UTR of NLRP3 mRNA, and the target relationship was identified by dual-luciferase reporter detection kit. RESULTSThe volume of KCs at 72 h was larger than that at 24 h, with clear boundary and stable shape. The result of carbon ink experiment showed that a large number of black particles were observed in the cells, which proved that the cells had strong phagocytic capacity and were KCs. After the KCs was induced by LPS at 1 mg/L, the level of miR-133 was decreased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were increased (P<0.05). After transfection with miR-133 inhibitor, the level of miR-133 in the cells was decreased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were increased (P<0.05). After transfection with miR-133 mimic, the level of miR-133 in the cells was increased, while the expression of NLRP3 at mRNA and protein levels, the caspase-1 protein in the cells, and the levels of IL-1β and TNF-α in cell culture medium were decreased (P<0.05). TargetScan analysis showed that the 3'UTR of NLRP3 mRNA contained the conservative bases of miR-133 sequence. Relative activity of luciferase in the cells transfected with miR-133 mimic was decreased (P<0.05). CONCLUSION miR-133 attenuates inflammation of mouse KCs by targeting NLRP3, thus protecting the KCs.     

Role of p38MAPK in production of pro-inflammatory cytokines in Kupffer cells from severe acute pancreatitis rats

AIM:To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in the Kupffer cells (KCs) production of pro-inflammatory cytokines, tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β), in severe acute pancreatitis (SAP) rats.METHODS:Sprague-Dwaley rats were randomized into three groups:①sham operation rats, ②SAP rats, ③SAP rats given the p38 MAPK inhibitor CNI-1493(10 mg/kg, iv). The SAP model was induced by the bili-pancreatic duct infusion with 5% sterile soduim taurocholate solution. Rats from each group were killed at 12 h after sham operation or SAP and Kupffer cells (KCs) were isolated. The mRNA expressions of TNF-α and IL-1β (by quantitative real-time RT-PCR) and p38 MAPK activity (by Western blot analysis) in KCs were examined. The levels of TNF-α and IL-1β in plasma were determined by ELISA.RESULTS:There was a significant acvitation of p38 MAPK in KCs harvested from SAP rats than those from sham operation rats. SAP also promoted the mRNA expressions of TNF-α and IL-1β in KCs and the plasma levels of TNF-α and IL-1β. These events were significantly inhibited by treatment with CNI-1493.CONCLUSIONS:p38 MAPK activation is one important aspect of the signaling events that may mediate the KCs production of pro-inflammatory cytokines, TNF-α and IL-1β, in SAP rats. The inhibition of the p38 MAPK may be a potential target in the prevention and treatment of SAP.     

Effect of antifibrotic herb serum on rat hepatic stellate cell proliferation and collagen synthesis

AIM: To investigate the effect of Chinese herbs, Ganxianfang(GXF), on rat hepatic stellate cells (HSC) proliferation and collagen synthesis. METHODS: Two types of herb serum, portal venous serum and circumferential venous serum, were prepared from rats infused intragastrically with 16, 8, 4 times adult dose of GXF decoction. HSC isolated from rat liver were processed with the above sera in vitro. Then we mensurated the radioactivity of HSC admixed with [[³H]H]proline and [[³H]H]thymine to judge the effect on proliferation and collagen synthesis of HSC. RESULTS: Both two types of serum collected 0.5, 1, 2 h after intragastrical infusion inhibited HSC proliferation (P＜0.05), and the serum collected 1 h after intragastrical infusion had the strongest effect (P＜0.05). Portal serum decreasea collagen synthesis (P＜0.05), but circumferential serum had no effect (P＞0.05). CONCLUSION: Inhibition of HSC proliferation and decrease of collagen synthesis may contribute to the GXF antifibrotic action.     

Effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion-induced early liver injury in rats

AIM：To explore the effect of intervention for mast cell function before reperfusion on intestinal ischemia-reperfusion (IR)-induced early liver injury. METHODS：Adult SD rats (n=35) were randomized into 5 groups with 7 rats each: sham operation group (S group), IR group, cromolyn sodium treatment group (IR+C group, 25 mg/kg), ketotifen treatment group (IR+K group, 1 mg/kg), compound 48/80 treatment group (IR+CP group, 0.75 mg/kg). IR was induced by superior mesenteric artery occlusion for 75 min followed by 4 h of reperfusion. The agents were intravenously administered 5 min before reperfusion. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and histamine, and the liver levels of lactate dehydrogenase (LDH), tumor necrosis factor α (TNF-α), interleukin-8 (IL-8), malondialdehyde (MDA) and superoxide dismutase (SOD) were assessed. The liver histopathologic changes were also evaluated. RESULTS：IR resulted in severe liver injury as demonstrated by great increases in injury scores, concomitant significant increases in serum levels of AST, ALT and histamine, and liver levels of LDH, TNF-α, IL-8, and MDA, accompanied by reduced SOD activity (all P<0.05 vs S group). Treatment with cromolyn sodium or ketotifen markedly alleviated IR-mediated liver injury as confirmed by significant reduction of the above biomedical changes, whereas compound 48/80 further aggravated liver injury by dramatically enhancing the biomedical changes (all P<0.05 vs IR group). CONCLUSION：Inhibition of mast cell function before reperfusion may reduce early liver injury induced by intestinal ischemia reperfusion. Histamine, oxidative stress and inflammatory response may provide promising effects on it.     

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.     

Apoptosis and its modulation in hepatic ischemia and reperfusion injury

Hepatic ischemia/reperfusion injury (HIRI) exists in lots of process of clinical pathology and operation. Apoptosis is an active process controlled by some gene and factors such as Fas/FasL, caspases and Bcl-2 families. More and more studies suggest that HIRI is associated with apoptosis. This article summarized the mechanisms and gene modulation of apoptosis during HIRI and the significance of suppressing apoptosis in preventing HIRI.     

Role of glucocorticoid receptor in hepatic injury in early stage after severe multiple injury

AIM: To investigate the functions of GR in the course of hepatic secondary injury after severe multiple injury. METHODS: Rat model was produced by adopting severe thoracic impact injury accompanied with mono-side femur fracture, and glucocorticoid receptor was blocked before severe multiple injury. Hepatic macropathology and alterations under light microscope were examined. Maximal binding volume of glucocorticoid receptor (GR) in hepatic tissue was assayed by radio-ligand binding assay and protein content was assayed by Western blot. RESULTS: Maximal binding volume and protein content of GR were gradually decreased in hepatic tissue after severe multiple injury, obviously lower than that in normal control at 4 h after trauma (P＜0. 01), and the lowest value was at 12 h after trauma. Maximal binding volume was 12. 9% of normal control (P＜0. 01), and protein content was 21. 9% of normal control (P＜0. 01). In addition, no significant pathological alteration in liver was found after trauma. However, the pathological analysis showed that the administration of GR blocker before severe multiple injury could cause severe hepatic congestion and infiltration of inflammatory cells in hepatic sinusoid in a dose-dependent manner. CONCLUSION: GR insufficiency could cause secondary hepatic injury in early stage after severe multiple injury, implying an important role of GR in injury and anti-injury mechanism of hepatic tissue cells.     

Expression of Th cytokines in peripheral blood before and after splenectomy in immune thrombocytopenia patients

AIM: To investigate the changes of Th cytokines before and after splenectomy in immune thrombocytopenia (ITP) patients. METHODS: The QuantiGene Plex method was used to measure the mRNA expression of Th1, Th2 (IL-4, IL-5, IL-6 and IL-10), Th3 (transforming growth factor β₁,TGF-β₁), and Th17 (IL-17) cytokines in peripheral blood of ITP patients before and after laparoscopic splenectomy and those in peripheral blood of healthy controls. RESULTS: The mRNA level of IL-2 was significantly decreased in ITP patients before operation compared with the healthy controls, whereas IL-17 was obviously over-expressed. No significant difference of the other cytokines between preoperative group and the normal controls was found. After splenectomy, the expression levels of both IL-2 and TGF-β₁ were significantly higher than those in preoperative group and the normal controls. IL-2 was also significantly increased after operation, but was still lower than that in the normal controls. No significant difference of other cytokines between postoperative group and healthy controls was observed. In addition, The Th1 cytokines (IL-2 and IFN-γ) were found to be positively correlated (r=0.647, P<0.01) in preoperative patients, while no correlation was found between the other cytokines. There was a positive correlation between IL-2 and IFN-γ (r=0.787, P<0.01) in postoperative patients. IL-17 also had positive correlations with IL-2 (r=0.554,P<0.01) and IFN-γ (r=0.461,P<0.05) in ITP patients after operation, respectively. CONCLUSION: There is an imbalance of Th cytokines in ITP patients. The mechanism of splenectomy for treating ITP may be associated with the balance regulation of Th cytokines.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Protective effect of emodin on lung injury induced by hepatic fibrosis in rats

AIM:To explore the protective effect of emodin on lung injury induced by hepatic fibrosis in rats. METHODS:The hepatic fibrosis rat model was established with multiple pathogenic factors (CCl 4, ethanol, high fat, high cholesterol and low choline) and treated with different doses (20 mg/kg and 40 mg/kg) of emodin for 4 weeks. The hepatic index was measured. The biochemical indexes, endotoxin, homocysteine, albumin, aspartate aminotransferase,alanine aminotransferase, total bilirubin, total cholesterol and triglyceride, and hepatic fibrosis indexes, hyaluronic acid, laminin, collagen IV and procollagen Ⅲ, were detected. The histopathological changes of the liver were observed. The pulmonary index was determined. The histopathological changes of the lungs were observed. The levels of tumor necrosis factor α (TNF-α), malondialdehyde (MDA), nitric oxide (NO) and peroxynitrite (ONOO-) in the lung tissues were analyzed. RESULTS:The rat hepatic fibrosis model was successfully established. In model group, lung edema and inflammation occurred, and the pulmonary index and the levels of TNF-α, MDA, NO and ONOO- in the lung tissues were increased significantly. In emodin treatment groups, the pulmonary indexes were lower than that in model group. The pathological injury of the lung tissues was alleviated. The levels of TNF-α, MDA, NO and ONOO- in the lung tissues were decreased. CONCLUSION:Emodin has a protective effect on lung injury induced by hepatic fibrosis in rats.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.     

Changes of aquaporin 4 expression after hepatic ischemia-reperfusion injury in rats

AIM: To study the relationship between the changes of aquaporin 4 (AQP4) expression and the liver functions in the process of hepatic ischemia-reperfusion (I/R) injury in rats. METHODS: Forty-eight Wistar rats were used to establish the animal model of hepatic I/R injury. The rats were subject to ischemia for 30 min and were randomly divided into 4 groups according to the time of reperfusion: 2 h group, 1 day group, 3 days group and 7 days group. The corresponding control animals were also set up. The serum was collected for detecting direct bilirubin (DB), indirect bilirubin (IB) and alanine transaminase (ALT). The pathological changes of the liver tissues were observed under microscope with HE staining. The protein expression of AQP4 was measured by the method of immunohistochemistry and the mRNA expression of AQP4 was detected by RT-PCR. RESULTS: Under microscope, degeneration and necrosis of the hepatic cells were observed in the liver tissues in I/R injury groups. Compared with sham operation group, the concentrations of DB, IB and ALT activity in I/R injury groups increased obviously, peaking on the first day after operation, then declining continuously and restoring to the normal levels on the 7th day after operation. The expression of AQP4 were significantly decreased in I/R injury animals in 2 h group, 1 day group and 3 days group, and reached the minimum level on the first day. The mRNA expression levels of AQP4 were also deceased in hepatic I/R injury rats in 2 h group, 1 day group and 3 days group, and reached the minimum level on the first day after operation, then increased slowly and restored to the normal levels on the 7th day after operation. CONCLUSION: Hepatic ischemia-reperfusion induces a decrease in AQP4 expression and impairs the liver functions, indicating an important role of AQP4 in the pathogenesis of liver ischemia-reperfusion injury.     

Effect of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells

AIM: To investigate the effects of siRNA-mediated Smad3 silence on proliferation and apoptosis in activated hepatic stellate cells (HSCs).METHODS: HSCs-T6 cells were divided into 3 groups: blank group, negative control group and siRNA-Smad3 transfection group. The siRNA-Smad3 was transfected into HSCs-T6 cells. At different time points after transfection, cell proliferation was measured by CCK-8, cell apoptosis was detected by flow cytometry, and protein levels of P53 and Bcl-2 were determined by immunocytochemistry.RESULTS: HSCs proliferation was significantly inhibited at the time points of 24 h, 48 h and 72 h after transfection. Meanwhile, the apoptosis of HSCs was significantly increased in siRNA-Smad3 transfection group (P<0.01). Compared to the control cells, the protein expression of P53 was significantly increased while Bcl-2 protein was significantly decreased 48 h after transfection in siRNA-Smad3 transfection group (P<0.01).CONCLUSION: The siRNA-mediated Smad3 silence significantly inhibits HSCs proliferation and induces apoptosis by up-regulating the P53 expression and down-regulating the Bcl-2 expression in HSCs.     

Lentiviral vector CV072-mediated Mfn2 over-expression inhibits hepatic stellate cell activation

AIM:To construct a lentiviral vector carrying mitofusin 2 (Mfn2), and to investigate the inhibitory effect of Mfn2 on the activation of rat hepatic stellate cells and its mechanism of reducing the formation of hepatic fibrosis-related factors. METHODS:The lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP containing Mfn2 was constructed and transfected into the hepatic stellate cells. The expression of green fluorescent protein was observed under fluorescence microscope, and the transfection efficiency was evaluated. The protein levels of Bax, Bcl-2, cleaved caspase-3, α-SMA, TGF-β1, Smad2 and Smad3 were detected by Western blot. The levels of type I collagen, type Ⅲ collagen and type IV collagen in the cell culture supernatants were determined by ELISA. RESULTS:Compared with control group, the apoptosis of the hepatic stellate cells transfected with lentivirus over-expression vector CV072-pCMV-Mfn2-EGFP was increased, and the protein levels of proapoptotic molecules Bax and cleaved caspase-3 were increased (P<0.01). TGF-β1/Smad pathway-related proteins TGF-β1, p-Smad2 and p-Smad3 were decreased, and the levels of fibrosis-related proteins α-SMA, type I collagen, type Ⅲ collagen and type IV collagen were decreased (P<0.01). CONCLUSION:Transfection of lentiviral over-expression vector CV072-pCMV-Mfn2-EGFP effectively inhibits hepatic stellate cell activation in vitro and may reduce the production of hepatic fibrosis-related factors by inhibiting TGF-β1/Smad pathway.