ABSTRACT
【Objective】 To observe the therapeutic effects of Axitinib, a tyrosine kinase receptor inhibitor, on liver fibrosis. 【Methods】 In vivo, CCL4 was injected intraperitoneally to induce liver fibrosis in mice. After modeling, Axitinib-carboxymethyl cellulose (Axitinib-CMC) solution or CMC solution were administered by gavage. After 2 weeks of modeling, 4 weeks of modeling, 1 week of treatment and 2 weeks of treatment, the mice were killed and their liver tissues were stained by HE, Masson and α - SMA immunohistochemistry. In vitro, human hepatic stellate cell line (LX2) and human normal liver cell line (LO2) were intervened with different concentrations of Axitinib-CMC solution. MTT assay was performed 48 h and 72 h after the intervention. Flow cytometry was used to observe the apoptosis of the two cell lines. Western blotting was used to detect the expressions of Fas, Caspase-8, Caspase-3 and Bcl-2 proteins. 【Results】 HE and Masson staining results showed that CCL4 could induce liver fibrosis in the mice, and the degree of liver fibrosis was more severe in the 4-week than that in the 2-week treatment group. After treatment with Axitinib, the collagen staining area and the positive expression level of α-SMA were significantly lower than those in 4-week group and CMC treatment control group (P<0.05). In vitro, Axitinib could effectively inhibit the viability of hepatic stellate cell line LX2 and promote its apoptosis. Meanwhile, the expressions of pro-apoptotic proteins Fas, Caspase-8 and Caspase-3 increased, while the expression of apoptosis suppressor gene Bcl-2 decreased. However, the above changes were not found in control hepatocyte line LO2. 【Conclusion】 Axitinib exerts an anti-fibrosis effect by inducing apoptosis of hepatic stellate cells, and has no significant effect on normal hepatocytes.
ABSTRACT
OBJECTIVE:To establish the method for simultaneous determinations of rutin,forsythin and platycodin D in Sangju ganmao pills.METHODS:HPLC-MS method was adopted.The determination was performed on Waters Atlantis C18 column with mobile phase consisted of acetonitrile-0.1％ formic acid (gradient elution) at the flow rate of 0.2 mL/min.The column temperature was set 35 ℃,and sample size was 10 μL.The ionization mode was electrospray ion,and the reaction mode was multi-reaction monitoring.By positive ion detection mode,the drying gas and nebuliser gas were all high purity nitrogen.The drying gas temperature was 270 ℃.The drying gas flow rate was 25 L/min.The sheath gas flow rate was 10 L/min.The capillary voltage was 4 500 V.The nozzle voltage was 2 000 V and the scanning time was 0.1 s.RESULTS:The linear range of rutin,forsythin and platycodin D were 0.010 82-2.164 μg/mL (r=0.999 7),0.010 18-2.036 μg/mL (r=0.999 4),0.010 27-2.054 μg/mL (r=0.999 7),respectively The limits of quantification were 1.250,0.260,2.720 ng/mL,and the limits of detection were 0.380,0.078,0.820 ng/mL.RSDs of precision,stability and reproducibility tests were all no more than 3.0％.The recoveries were 97.88％-99.88％ (RSD=0.72％,n=6),98.48％-103.13％ (RSD=1.91％,n=6),98.79％-101.41％ (RSD=1.05％,n=6).CONCLUSIONS:This method is simple,precise,stable and reproducible,and can be used for simultaneous determination of rutin,forsythin and platycodin D in Sangju ganmao pills.