Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.     

Upregulation of proteasome activity by 18α-GA promotes proliferation of late-passage BMSCs in vitro

AIM: To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells (BMSCs). METHODS: Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity. The expression of senescence-related proteins p53, p21 and p16 was detected by senescence-associated β-galactosidase (SA-β-Gal) staining and Western blot. The cell proliferation, the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK-8 assay, BrdU incorporation, Western blot and flow cytometry. RESULTS: Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01). SA-β-Gal-positive cells in 18α-GA group decreased, and cell staining was lighter. The contents of p53 and p21 in 18α-GA group were decreased (P<0.05). The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01). BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group (P<0.05). The cells in G₁ phase in 18α-GA group decreased significantly compared with DMSO group, while the cells in S phase increased significantly (P<0.05). The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05). CONCLUSION: Activation of the proteasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up-regulation of the cell cycle-related proteins.     

Donor age affects confluent EPCs on phenotypic transition,proliferation and migration of smooth muscle cells

AIM: To explore the effects of confluent endothelial progenitor cells (EPCs) derived from young and aged rats on the phenotype conversion, proliferation and migration of vascular smooth muscle cells (SMCs). METHODS: Mononuclear cells were obtained from the bone marrow of young (1~2 month old) and aged (19 to 26 month old) Sprague-Dawley rats and cultured with medium DMEM/F12 (containing 15% fetal bovine serum, endothelial cell growth supplements (ECGs) 100 g/L, 1×10⁵ units/L of penicillin and streptomycin, respectively). EPCs were characterized as double positive for DiI-Ac-LDL uptake and lectin binding. Abdominal aorta was obtained from 1 to 2 month old Sprague-Dawley rats. Vascular SMCs were cultured by tissue explant method and identified by α-SM-actin immunofluorescence. In transwell co-culture system, the confluent EPCs located in the upper chamber and SMCs were seeded on the lower chamber. The experiments were divided into passage 3 SMCs group (P3), passage 4 SMCs group (P4), passage 4 SMCs co-culture with EPCs derived from young rats group (P4YE) and passage 4 SMCs co-culture with EPCs derived from aged rats group (P4AE). The protein expression of α-SM-actin and osteopontin was detected by Western blotting. [³H]-TdR incorporation assay was used to determine the proliferation. SMC migration was analyzed by scratch wound healing assay. RESULTS: Compared with P3 group, α-SM-actin expression in P4 group significantly decreased and osteopontin protein expression obviously increased, whereas no significant change was found in P4YE group. Compared with P4 group, confluent EPCs derived from young and aged rats both markedly increased α-SM-actin and decreased osteopontin expression in P4 SMCs. Compared with aged rat-derived EPCs, young rat-derived EPCs were more effectively to induce a delayed SMC phenotype transition (from contractile phenotype to a synthetic phenotype), and to inhibit SMC proliferation and migration. CONCLUSION: Co-culture of confluent EPC induces a delayed vascular SMC phenotype transition and inhibits SMCs proliferation and migration. Young rat derived EPCs are more effective to induce a delayed vascular SMC phenotype transition and has stronger inhibitory effects on SMCs proliferation and migration compared with that derived from aged rats.     

Activation of Nrf2 by 18α-GA affects proliferation of adult neural stem cells

AIM: To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) activation by 18α-glycyrrhetinic acid (18α-GA) on the proliferation and self-renewal of adult neural stem cells (aNSCs), and to explore an effective way of maintaining the viability of aNSCs. METHODS: NSCs were dissociated from subventricular zone of the mice at postnatal days 0, 60, and 300. The expression levels of Nrf2 in the NSCs at various ages were compared. After treatment with 18α-GA, the expression of Nrf2 was examined by real-time PCR and Western blot. shRNA lentiviral vector (LV) carrying green fluorescent protein (GFP) gene was constructed to knock down Nrf2 expression. The knockdown efficiency in the aNSCs was detected by real-time PCR and Western blot. Subsequently, the aNSCs were divided into DMSO group, 18α-GA group, LV-GFP group and LV-Nrf2-shRNA group. BrdU incorporation assay, Tuj1 staining, CCK-8 assay, Hoechst 33342/PI staining and detection of reactive oxygen species (ROS) were performed to analyze the proliferation, differentiation, viability, apoptosis and oxidative stress levels of the NSCs. RESULTS: The mRNA expression level of Nrf2 in adult and aged NSCs was significantly lower than that in newborn NSCs (P<0.01), while the ROS level of aNSCs was significantly higher (P<0.05). After treatment with 18α-GA, the expression level of Nrf2 in the aNSCs was significantly up-regulated as compared with DMSO group (P<0.01). Increased number of BrdU⁺ and Tuj1⁺ cells was observed in 18α-GA group, indicating that 18α-GA-treated cells had higher viability (P<0.05). Meanwhile, there were fewer apoptotic cells and lower ROS level in 18α-GA group than those in DMSO group (P<0.05). After knockdown of Nrf2 in aNSCs and then treated with 18α-GA, there were less BrdU⁺ and Tuj1⁺ cells, as well as the aNSCs with lower viability in LV-Nrf2-shRNA group (P<0.05). Moreover, the ROS level was increased in LV-Nrf2-shRNA group as compared with LV-GFP group (P<0.05). CONCLUSION: Activation of Nrf2 by 18α-GA elevates the antioxidant capacity of aNSCs, thus ameliorating the cell proliferation and differentiation potentials.     

Vascular endothelial growth factor on function of endothelial progenitor cells from peripheral blood

AIM: To investigate the effect of vascular endothelial growth factor(VEGF) on the biological function of peripheral endothelial progenitor cells (EPCs) and to find the suitable concentration to promote the growth of EPCs.METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After culture for 4 days,attached cells were incubated with VEGF in a series of concentrations (0,10,20 and 50 μg/L) for 72 h,then attached cells were characterized with immunohistochemistry.EPC proliferation and migration activity were assayed with MTT assay and modified Boyden chamber assay,respectively.EPC adhesion assay was performed by replating MNCs on fibronectin-coated dishes,and then the adherent cells were counted.RESULTS: The EPCs from MNCs were successfully isolated and were differentiated to endothelial cells (ECs).Incubation of isolated human MNCs with VEGF increased the proliferative,migratory and adhesive capacities of EPCs,and this effect was most prominent when the concentration of VEGF was 20 μg/L after 72 hours.At the same concentration of VEGF,the functions of EPCs from patients with masculine coronary arteriography were lower than those of EPCs from patients with negative coronary arteriography.CONCLUSION: Functional activities of EPCs are decreased in patients with masculine coronary arteriography.The results suggest that the low concentration of VEGF may improve functional activities of EPCs.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.     

Nerve growth factor promotes proliferation of hepatocytes in vitro via TrkANGFR signal pathway

AIM: To observe the expression and distribution of nerve growth factor (NGF), its high-affinity tyrosine kinase A receptor (TrkA^NGFR) and low-affinity p75 neurotrophin receptor (p75^NTR) in hepatocytes(L02 cells), and to investigate the effects of exogenous recombinant human NGF-β on L02 cells. METHODS: L02 cell line was used in the experiment. The expression and intracellular distribution of NGF, TrkA^NGFR and p75^NTR were detected by the methods of immunocytochemistry and fluorescent quantitative PCR. The proliferation of L02 cells after exposed to exogenous NGF-β, anti-NGF, anti-TrkA^NGFR and anti-p75^NTR was detected by XTT {2,3-bis(2-methoxy-4-nitro-5- sulfophenyl)-5- -2H-tetrazolium hydroxide} assay. The cell apoptosis and the change of cell cycle of L02 cells after exposed to exogenous NGF-β were determined by flow cytometry with Annexin V-FITC and PI staining. RESULTS: The expression of NGF was mainly localized in the cytoplasm and nuclei of L02 cells. The expression of TrkA^NGFR was highly and the expression of p75^NTR was weakly detected in the cell membrane and cytoplasm of L02 cells. Exogenous NGF-β promoted the expression of NGF and TrkA^NGFR in L02 cells. Low dose of exogenous NGF-β (12.5-200 μg/L) promoted L02 cell proliferation and inhibited the cell apoptosis by affecting the cell cycle in S-phase. High dose of NGF-β (>400 μg/L) did not promote L02 cell proliferation. Specific neutralizing antibodies of NGF and TrkA^NGFR decreased the NGF-β-induced proliferation of L02 cells. However, blocking the binding of NGF to p75^NTR by anti-p75^NTR did not affect the NGF-β-induced proliferation of L02 cells. CONCLUSION: L02 cells express NGF, TrkA^NGFR and p75^NTR. Exogenous recombinant human NGF-β at optimal dose promotes L02 cell proliferation in a dose-dependent manner possibly via NGF/TrkA^NGFR signal pathway.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Effects of SRSF9/SRp30c on proliferation and migration abilities of glioma cells by regulating GRβ

AIM: To investigate the expression of serine-arginine-rich splicing factor 9/serine-arginine-rich protein 30c (SRSF9/SRp30c) and glucocorticoid receptor β (GRβ) in the glioma cells and the relationship of them. METHODS: Small interfering RNA (siRNA) was used to knock down the expression of SRSF9 in the U87 cells. Short hairpin RNA (shRNA) derived from lentivirus was used to establish U87 stable knockdown cell line. Fluorescence microscopy was used to observe and detect transfection efficiency. The expression of GRβ and SRSF9/SRp30c at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability, colony formation ability and migration ability were measured by CCK-8 assay, colony formation assay and wound healing experiment. RESULTS: The mRNA and protein levels of SRSF9/SRp30c and GRβ in the U87 cells were both down-regulated after knockdown of SRSF9 (P<0.05). Fluorescence microscopic observation showed that a stable cell line was constructed successfully, and the transfection efficiency exceeded 80%. After knockdown of SRSF9 expression in the U87 cells, the cell viability and colony formation ability were reduced (P<0.05). The migration ability was weakened significantly after SRSF9 was knocked down (P<0.05). CONCLUSION: SRSF9/SRp30c may promote the proliferation and migration of the glioma cells by regulating GRβ.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Effect of annexin A2 on proliferation,migration and apoptosis of human cervical cancer HeLa cells

AIM：To explore the effect of annexin A2 (ANXA2) on the proliferation, migration and apoptosis abilities of human cervical cancer HeLa cells. METHODS：Overexpression vectors and siRNA of ANXA2 were constructed, and then transfected into HeLa cells. The HeLa cells were divided into 4 groups:control group, scramble group, ANXA2 overexpression group and ANXA2-siRNA group. The expression of ANXA2 at mRNA and protein levels was examined by real-time PCR and Western blotting. MTT assay, Boyden chamber and flow cytometry were used to determine the effect of ANXA2 on the proliferation, migration and apoptosis abilities of the HeLa cells. RESULTS：The proliferation and migration of HeLa cells were obviously promoted by ANXA2 overexpression. The proliferation and migration of HeLa cells were remarkably inhibited by the transfection of ANXA2-siRNA. ANXA2 had no effect on apoptosis of HeLa cells. CONCLUSION:Silencing of ANXA2 effectively inhibits the proliferation and migration of cervical cancer cells, but has little effect on apoptosis. ANXA2 may play a pivotal role in the occurrence and development of cervical cancer, and may be used as a molecular target for the treatment of cervical cancer.     

Activation of α7nAChR promotes wound healing in diabetic mice by suppressing TNF-α expression

AIM: To explore the role of α7 nicotinic acetylcholine receptor (α7nAChR)-specific agonist PNU-282987 in promoting wound healing in diabetic mice by suppressing the expression of tumor necrosis factor α （TNF-α）.METHODS: A model of incised wound was established in diabetic mice or normoglycaemic mice (control). Skin samples were taken on 1 d, 3 d, 5 d, 10 d, 14 d and 21 d post-injury (5 mice in each posttraumatic interval). The numbers of macrophages and fibroblasts, the expression of TNF-α and the deposition of collagen were detected by the methods of immunohistochemistry, Western blotting and Masson staining, respectively. After incised wound was performed in the diabetic mice, PNU-282987 was applied by intraperitoneal injection at suitable posttraumatic interval. The above indexes were investigated again.RESULTS: Compared with control group, the diabetic mice presented delayed wound healing. In diabe-tic mice, the infiltration of macrophages and the expression of TNF-α were significantly reduced in the early phase during wound healing, while they were significantly increased from 5 d post-injury. Besides, from 5 d to 21 d post-injury, the wounds in diabetic mice showed decreased number of fibroblasts and deposition of collagen. From 5 d post-injury, PNU-282987 was applied to diabetic mice, which significantly down-regulated the expression of TNF-α, and increased the number of fibroblasts and the content of collagen in the wounds, eventually promoted wound healing.CONCLUSION: Inflammatory reactions delay wound healing in diabetic mice. Activation of α7nAChR promotes wound healing in diabetic mice by suppressing the expression of TNF-α.     

Effects of SSeCKS on adhesion and migration of bovine pulmonary artery endothelial cells stimulated by fibronectin

AIM: To study the effects of Src suppressed C kinase substrates(SSeCKS) on the adhesion and migration of bovine pulmonary artery endothelial cells (BPAECs) stimulated by fibronectin(FN).METHODS: Cultured BPAEC were stimulated by different concentrations of FN.SSeCKS expressions were detected by Western blotting.Cultured BPAECs were treated with Ro31-8220 and calphostin C (a PKC inhibitor).The locations of SSeCKS,F-actin and vinculin before and after stimulation were observed by confocal microscopy.RESULTS: After stimulated by FN,SSeCKS expression in BPAECs increased in concentration and time dependent manners.After treated with Ro31-8220 and calphostin C,the adhesion and migration of BPVECs were restrained,the expression of SSeCKS was inhibited,SSeCKS distributed from cytosol to perinuclear,and the colocalization of SSeCKS with F-actin,vinculin at the edge of BPAECs was reduced.CONCLUSION: SSeCKS may play an important role in ECs adhesion and migration stimulated by fibronectin.The process can be inhibited by Ro31-8220 and calphostin C.     

Regulatory effect of astragaloside IV on SDF-1α/CXCR4 in EPCs

AIM: To investigate the effects of astragaloside IV (AS-IV) on chemokine receptor 4 (CXCR4) and stromal cell-derived factor 1α (SDF-1α) in endothelial progenitor cells (EPCs) and its mechanism. METHODS: Rat bone marrow-derived EPCs were cultured in vitro. The proliferation, adhesion, migration, apoptosis and tube formation capacity of EPCs treated with AS-IV and AMD3100, a specific blocker of CXCR4, were observed. The effects of AS-IV on the expression of SDF-1α/CXCR4 at mRNA and protein levels and the protein level of p-CXCR4 in the EPCs were determined. RESULTS: AS-IV significantly enhanced the proliferation, adhesion, migration and tube formation abilities of EPCs, reduced the apoptosis of EPCs, and up-regulated the mRNA and protein expression of SDF-1α and CXCR4 and the p-CXCR4 protein level in the EPCs. On the other hand, AMD3100 blocked the up-regulating effect of AS-IV on the mRNA and protein expression of CXCR4 and the p-CXCR4 protein level in the EPCs, but did not affect the effect of AS-IV on the expression of SDF-1α. CONCLUSION: AS-IV might enhance the biological function of EPCs by regulating the expression of SDF-1α/CXCR in EPCs.     

Effect of plumbagin on levels of Nox4/ROS and α-SMA in human hepatic stellate cells

AIM: To observe the effect of plumbagin on the mRNA and protein expression of nicotinamide adenine dinucleotidephosphate oxidase 4 (Nox4), reactive oxygen species (ROS) level and protein expression of α-smooth muscle actin (α-SMA) in the HSC-LX2 cells stimulated with transforming growth factor β1 (TGF-β1) in vitro. METHODS: HSC-LX2 cells were cultured in vitro and divided into blank group, model group, high-, medium- and low-dose (2, 1.5 and 1 μmol/L) plumbagin groups. After incubated with each drug for 72 h, the mRNA expression of Nox4 was detected by RT-PCR. ROS levels were tested by in situ loading probe method. The protein contents of Nox4 and α-SMA were measured by Western blot. RESULTS: Compared with model group, after treated with plumbagin for 72 h, the mRNA expression of Nox4, ROS level and α-SMA protein were significantly decreased in high-and medium-dose plumbagin groups (P<0.01). CONCLUSION: Plumbagin inhibits the activation of HSC-LX2 cells via decreasing the expression of Nox4, thus decreasing ROS levels.