OBJECTIVE:To study inhibitory effect of Lycianthes biflora polysaccharide on the proliferation of hepatic stellate HSCs-T6 cells in rats and its mechanism. METHODS:After treated with 0(blank control),50,100,200μg/ml L. biflora polysac-charide for 24 and 48 h,the activity of hepatic stellate HSCs-T6 cells in rats was determined by MTT assay and inhibitory rate of cell proliferation was calculated;the content of Hyp in supernatant was detected by immunohistochemical assay. After 48 h,the ex-pression of cellular α-smooth muscle actin(α-SMA)was measured by immunohistochemical assay;both transforming growth fac-tor β1(TGF-β1)and Smad3 were measured by Western blot assay. RESULTS:Compared to blank control,50,100 and 200 μg/ml L. biflora polysaccharide could inhibit the proliferation of HSCs-T6 cells;cell inhibitory rates were 39.84%-69.31% and 45.16%-82.93% respectively after treated for 24 and 48 h,which were positively associated with time and concentration. The con-tents of Hyp in supernatant were 178.36-93.25 μg/ml and 131.94-68.74 μg/ml respectively after treated with different concentrations of PRP for 24 and 48 h,which were negatively associated with time and concentration. The protein level of TGF-β1 and Smad3 de-creased after treated with L. biflora polysaccharide for 48 h(P<0.01). CONCLUSIONS:L. biflora polysaccharide can inhibit the proliferation of hepatic stellate HSCs-T6 cells in rats,and its mechanism is associated with the inactivation of endogenous TGF-βpathway for reducing collagen production.

Objective To study the effect and mechanism of Hedyotic diffusa injection in reversing multi-drug resistance of BEL-7402/5-FU cells.Methods The IC50 of BEL-7402/5-FU cells was examined by MTT,apoptosis was detected by flow cytometry,and the content of tyrosine kinase in total protein of tumor cells was detected by enzyme-linked immunosorbent assay (ELISA).Results The IC50 of Hedyotic diffusa injection (6 μL/mL ) combined with 5-FU (636.5 μmol/L)decreased significantly compared with that of 5-FU applied alone (1 071.6 6μmol/L). The reversal on BEL-7402/5-FU was 1.68-fold.After injection of 5-FU plus Hedyotic diffusa ,the apoptosis rate of BEL-7402/5-FU cells was increased significantly and the tyrosine kinases of BEL-7402/5-FU cells decreased significantly.Conclusion Hedyotic diffusa injection plays a certain role in reversing multi-drug resistance of BEL-7402/5-FU cells,which may be related to decreasing their tyrosine kinases.

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.

Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal en-ables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concen-tration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3％and 110.8％with relative standard deviations of 7.3％-17.6％,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.

[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.