| Objectives:1) To confirm the therapeutic and anti-viral effects of baicalin in the murine model of MCMV hepatitis;2) To explore the effects of baicalin inducing anti-MCMV immunity and its possible links and mechanisms.Methods:1) The hepatotoxic experiment of baicalin:According to the medium dosage of baicalin for adults, we calculate the dosage for mice is about 60mg/kg. The doses gradient designed by double increase were 60mg/kg,120 mg/kg,240 mg/kg and 480 mg/kg. The normal mice were injected with different doses of baicalin intraperitoneal once a day and total for 14 days. Then detect the level of serum alanine aminotransferase (sALT) and observe the pathological changes of livers, in order to evaluate the hepatotoxic effects of baicalin;2) The establishment of the animal model:According to the method of XuYi, BALB/c mice of 4.5 weeks old were intraperitoneal injected with the MCMV Smith strain, which was propagated in the immunosuppressive mice to increase the virulence. Thus, MCMV disseminated infection model was established. Baicalin of 60mg/kg was injected intraperitoneal 24 hours after the inoculation of MCMV, once a day and total for 14 days. The placebo group and normal control group were established simultaneously;3) The estimation of liver pathological changes:Liver tissue lesions were observed on 3,7, and 14 days after giving drugs. The pathological changes were evaluated by the hepatic active integral (HAI), and the changes of liver cell function by the level of sALT; 4) The assessment of virology: The infectious virus in the salivary gland was detected by the inhibitive plaque assay on 14 days of baicalin treatment, and compared with that of the placebo group;5) The expression of Thl/Th2-type cytokines: Murine spleen cells suspension was prepared on day 3,7 and 14. Then, cultured for 48h supplemented with ConA and inactivated MCMV in vitro. The supernatant was collected, and the level of IFN-gamma (Thl-type cytokines) was measured by Double antibody sandwich ELISA. A control without stimulus was established. Meanwhile the level of serum IL-4 (Th2-type cytokines) was also detected;6) The expression of transcription factor TBX21 and GATA-3 protein specific for Thl and Th2 cell respectively:Protein was extracted from spleen tissue. The protein expression of TBX21 and GATA-3 specific for Th1 and Th2 cell was detected by Western blot with internal control, and semi-quantitatively analysed by the regulated K value.Results:1) The hepatotoxic effects of baicalin: There was no murine death and other abnormal response during the 14 days using intraperitoneal injection with baicalin of 60mg/kg,120 mg/kg,240 mg/kg and 480 mg/kg. The level of sALT in each group had no obvious difference compared with normal controls, and there had no significant pathological change on liver slices;2) The change of liver function:Compared with normal control, the level of ALT of placebo group increased significantly on day 3, reached the peak on day 7 and declined on day 14. Compared with the placebo group, the level of sALT of baicalin-treated group decreased evidently on day 3, and dropped further on day 7 and nearly to normal on day 14;3) The pathologic change of liver tissue: The HAI of placebo group reached the peak on day 3, then decrease gradually. The HAI of baicalin-treated group also reached the peak day 3, but liver damage was obviously less than that of placebo. The liver damage also relieved gradually and the mean of HAI was obviously lower than that of the placebo group on day 7 and 14;4) The variation of infectious virus in salivary glands:After baicalin treatment of 14 days, the infectious virus in the murine salivary glands was obviously lower compared with the placebo group;5) The expression of Thl-type cytokines:IFN-gamma in splenic cell supernatant reached the peak on day 7 both in baicalin-treated group and placebo one. They were all higher than those of normal control on days 3,7 and 14, and there were significant statistic differences. Compared with placebo group, the IFN-gamma level of baicalin-treated was significantly higher on day 3 and 7, but on dayl4, it was obviously lower than that of placebo group;6) The expression of Th2-type cytokines:The level of serum IL-4 of placebo group was remarkably lower than that of normal control on day 3, then elevated to the normal level on day 7, and was obviously higher than that of normal control on day 14. As to baicalin-treated group, its sIL-4 was also significantly lower than that of normal control on day 3, and reached the peak on day 7, which was little higher than that of normal control, but obviously higher than that of placebo group. And on day 14, the level decreased a little, which was markedly higher than that of normal control, but similar to that of placebo group;7) The expression of TBX21 protein:The expression of TBX21 protein of placebo group was little lower than that of normal control on day 3; but obviously higher on day 7 and 14. On day 3, the expression of TBX21 protein of baicalin-treated group was significantly higher than that of normal control and placebo group, it reached the peak on day 7, and decrease a little on day 14, which was markedly higher than that of normal control, but nearly equivalent to that of placebo group;8) The expression of GATA-3 protein: The expression of GATA-3 protein of placebo group and baicalin-treated group was obviously lower than that of normal control on day 3; however, on day 7 and 14, both were similar to that of normal control, and there was no significant difference between baicalin-treated group and the placebo one.Conclusion:1) Baicalin of 8 times to the medium doses has no side-effects to mouse liver tissue, which demonstrate that it is a safe Chinese medicine;2) In vivo, baicalin can improve the liver pathological damage caused by MCMV infection significantly, promote sALT back to normal, and reduce the infectious virus in salivary gland. All of those show that baicalin has the effect against cytomegalovirus in vivo, and therapeutic effect on MCMV hepatitis;3) During the early period of acute MCMV infection, mice mainly switch on the Th1-mediated cell immunity to remove the virus quickly;4) During the early period of acute MCMV infection, baicalin can promote Thl cell immunity by increasing the expression of TBX21 protein, so as to clear the virus and moderate the change of Th1/Th2 cell net caused by MCMV.
|
PDF Full Text Request