Objective To explore the protective effect and mechanism of vitamin D combined with puerarin on liver fibrosis.Methods The rats were divided into normal control group (C),tetrachloromethone group (CCl4),vitamin D group (V),puerarin group(P) and vitamin D combined with puerarin group(V+P).After 8 weeks,the rats were sacrificed and blood and liver samples were collected.The level of blood hyaluronic acid(HA) was tested.The hydroxyproline(Hyp) level in the liver was measured.The liver paraffin sections were made and examined by the sirius red staining.The mRNA levels of collagen Ⅰ and collagen Ⅲ in the liver tissue were detected by RT-PCR,and the levels of NF-κB and TNF-α in the liver were detected by Western blot.Results The CCl4 group appeared obvious liver fibrosis.The liver fibrosis degree was significantly improved in the group V,P and V+P,the blood HA level and liver Hyp level were reduced.The mRNA levels of collagen Ⅰ and collagen Ⅲ as well as the protein levels of NF-κB and TNF-α in the liver were significantly decreased.Among them.The liver fibrosis improvement degree in the V+P group was most significant.Conclusion Vitamin D combined with puerarin can protect rat liver fibrosis induced by CCl4 and its mechanism may be related with reducing the activation of hepatic stellate cells(HSC) and decreasing the collagenous fibers secretion.

Objective To investigate the effect of miRNA-193b (miR-193b) up-regulation on the apoptosis,invasion and metastasis of hepatitis B virus (HBV)-positive hepatocellular cells.Methods HepG2.2.15cells were cultured until logarithmic growth phase and transfected with miR-193b mimic at a concentration of 25,50,100 pmol.The control group was transfected with blank mimic (0 pmol).After 48 hours,the expression of miR-193b in each group was detected by real-time quantitative polymerase chain reaction (qRT-PCR),the apoptosis rate in each group was detected by flow cytometry,the cell migration was detected by cell scratch assay,the cell invasion was detected by Transwell assay,and the expressions of Bax,Bcl-2,matrix metalloprotease (MMP)-9 and MMP-2 protein were detected by Western blotting.Results qRT-PCR results showed that the miR-193b expressions of miR-193b mimic (25,50 and 100 pmol) groups and control group in HepG2.2.15 cells were 1.05 ±0.09,1.53 ±0.12,2.08 ±0.17 and 0.49 ±0.12,and the difference was statistically significant (F =261.35,P ＜ 0.001).Compared with the control group,miR-193b expression significantly increased in each transfection group (P =0.036;P =0.029;P =0.022).Flow cytometry results showed the apoptosis rates of miR-193b mimic (25,50 and 100 pmol) groups and control group were (30.28 ±5.22) ％,(53.41 ±6.18)％,(79.89 ±7.13)％ and (1.02 ±0.13)％,and the difference was statistically significant (F =357.19,P ＜ 0.001).Compared with the control group,the apoptosis rate significantly increased in each transfection group (P =0.025;P =0.010;P =0.007).Cell scratch assay results showed that the migration distances of miR-193b mimic (25,50 and 100 pmol) groups and control group were (27.53 ± 1.54) mm,(19.24 ± 2.12) mm,(13.42 ± 1.53) mm and (34.95 ± 1.92) mm,and the difference was statistically significant (F =408.62,P ＜ 0.001).Compared with the control group,the migration distance significantly decreased in each transfection group (P =0.032;P =0.007;P =0.006).Transwell experimental results showed that the absorbance values of miR-193b mimic (25,50 and 100 pmol) groups and control group were 1.02 ±0.12,0.59 ±0.13,0.42 ±0.10 and 1.68 ±0.16,and the difference was statistically significant (F =511.68,P ＜ 0.001).Compared with the control group,the cell invasion ability significantly weakened in each transfection group (P =0.028;P =0.005;P =0.002).Western blotting results showed that the expressions of Bax,Bcl-2,MMP-9 and MMP-2 protein of miR-193b mimic (25,50 and 100 pmol) groups and control group had statistically significant difference (F =264.38,P ＜ 0.001;F =437.19,P ＜ 0.001;F =377.46,P ＜0.001;F =208.79,P ＜ 0.001).Further paired comparison showed that the expressions of Bax,Bcl-2,MMP-9 and MMP-2 protein between mimics groups and control group were statistically significant (all P ＜0.05).Conclusion miR-193b can induce the apoptosis and inhibit the invasion,metastasis of HBV-positive hepatoeellular cells,and the mechanism may be related to the regulation of Bax,Bcl-2,MMP-9 and MMP-2 protein expression.

[Abstract] Objective: To investigate the miRNAs that can intervene Mcl-1 expression in HBV-related liver cancers and to study their synergistic anti-cancer effect with sorafenib. Methods: The expressions of miR-29, miR-101 and miR-193b in HepG2.2.15 (HBV positive) and HepG2.vc (HBV negative) cells were detected by qPCR. miRNA mimics of low expressed genes in HepG2.2.15 cells were synthesized and transfected into HepG2.2.15 and HepG2.vc cells, respectively. qPCR was used to detect target miRNA expression. Western blotting was used to detect the expression of mcl-1 protein in cells before and after transfection.At the same time, (1×10-9)~(1× 10-3) mol/L of sorafenib was add to both transfected and non-transfected HepG2.2.15 and HepG2.vc cells; 72 h later, the IC50 and cell apoptosis was evaluated. Results: The expression of miR-193b in HepG2.2.15 cells was significantly lower than that in HepG2.2.15 cells (P <0.05). The expression of miR-193b in HepG2.2.15 cells and HepG2.2.15 cells was significantly higher after miR-193b mimics transfection (P <0.05). Compared with HepG2.vc cells, the expression of Mcl-1 protein in HepG2.2.15 cells was significantly increased (P <0.05). The expression of Mcl-1 protein in HepG2.2.15 and HepG2.vc cells was significantly decreased after miR-193b mimics transfection (P<0.05). After miR-193b mimics transfection, sorafenib could significantly increase apoptosis rate of both HepG2.2.15 and HepG2.vc cells. Conclusion: The low susceptibility of HBV-related liver cancer to sorafenib may be related with the low expression of miR-193b in cancer cells. Mcl-1 might be used as a target of miR-193b, and miR-193b mimics have a significant synergistic effect with sorafenib.