Effect Of Solution Of Qingkailing On Immunological System Of Mouse And Studies On Inhibitory Effect Of QKL On HIV-1 In Vitro

Chapter 1 Effect of Solution of qingkailing on immunological system of mouse in vitroAim: To investigate the effect of Solution of qingkailing (QKL) on the activation, proliferation and cell cycle distribution oft lymphocytes, and on phagocytosis and NO production of peritoneal macrophage, and on apoptosis of thymocytes of mouse in vitro, in order to explore its effect on immunological system of mouse and its action mechanisms.Methods: Fluorescence conjugated monoclonal antibodies and flow cytometry were used to detect the expression of CD69 on T lymphocytes activated by ConA or not to analyze its effect on activation. MTT and WST-1 were used to examine the proliferation of T lymphocytes in addition to CFDA-SE staining together with flow cytometry. The distribution of the cell cycle was analyzed by PI staining together with flow cytometry. E. Coli stained by CFDA-SE and flow cytometry was utilized to observe its effect on macrophagic phagocytosis. NO production from macrophage activated by LPS was detected by Griess kit. Spontaneous apoptosis and apoptosis induced by DEX of thymocytes were detected by PI staining together with flow cytometry.Results: QKL had no evident influence on activation, proliferation and distribution of cell cycle of T lymphocytes without ConA. The expression rate of CD69 in activated T cells in response to ConA was (63.12±3.07)ï¼…. There was no evident difference of 1/5120 QKL with ConA. 1/1280,1/320,1/80 QKL reduced the expression rate of CD69. The number was (56.24±2.43)ï¼…,(51.89±2.77)ï¼…and (39.09±1.39)ï¼…respectly. QKL inhibited the proliferation of T lymphocytes activated by ConA. MTT and WST-1 assay in addition to CFDA-SE staining showed that 1/5120 QKL decreased the value of OD or PI. 1/2560 QKL significantly inhibited the proliferation. The number of EC₅₀ was 1/405.34,1/551.14 and 1/435.73 respectly. Flow cytometry analyse of cell cycle distribution revealed that QKL increased the number of S and G2/M phase and decreased the number of G0/G1 phase. However 1/160 QKL increased the number of Sub-G1 phase significantly. QKL inhibited the macrophagic phagocytosis significantly. There were remarkable difference of the percentage of phagocytic cells between control group and QKL groups. The number of control group was (61.33±2.45)ï¼…, and 1/2700,1/900,1/300,1/100 QKL was (57.62±1.67)ï¼…,(53.55±2.30)ï¼…,(48.92±2.01)ï¼…and (39.32±0.68)ï¼…respectly. QKL also reduced NO production from peritoneal macrophage activated by LPS. PI staining analysis the apoptotic peak showed the QKL inhibited significantly the spontaneous apoptosis and apoptosis induced by DEX of thymocytes.Conclusion: QKL has regulative effect on immunological function of mouse in many aspects. QKL inhibited significantly the activation and proliferation of T lymphocytes induced by ConA, and phagocytosis and NO production of peritoneal macrophage. In addition, QKL resisted the apoptosis ofthymocytes. That indicated QKL has evident immune suppressive effect. It inhibited specific cellular immune response and non-specific immune response.Chapter 2 Effect of QKL on mouse with normal situation, DTH and sepsisObjectives: To investigate the effect of QKL on mouse with normal situation, DTH and sepsis, in order to elucidate its inhibitory effect on immunological system in vitro coordinated in vivo and to conjecture its machenisms.Methods: QKL was injected hypodermicly for five days. By comparing the body weight, immune organs index and response of lymphocytes to ConA, to analyze the effect of QKL on immunological system of normal mouse. Delayed type hypersensitivity (DTH) mouse model was established by stimulation with dinitrofluorobenzene (DNFB). By comparing spleen index and swelling rate of pathogenetic ear, to illuminate the immunoregulation of QKL in vivo. Polymicrobial sepsis mouse model was induced by cecal ligation and puncture (CLP). The cumulative survival rate was observed in QKL and NS groups.Results: QKL had no influence on body weight and spleen index of normal mouse, but it could significantly decrease thymus index and inhibit the immune response of lymphocytes to ConA. QKL administration could lessen the swelling of pathogenetic ear of DTH mouse and decrease spleen index. QKL administration raised the mortality of mouse with sepsis.Conclusion: QKL can inhibit the immunological system of mouse with normal situation and DTH. QKL has no therapeutical effect on sepsis. In contrast, it improve the death of mouse with sepsis. Inhibiting the funcitons of T lymphocytes and macrophage may be the mechanisms.Chapter 3 Inhibitory effect of Solution of qingkailing and its drug serum on HIV-1 in vitroObjectives: To investigate the inhibitory effect of QKL and its drug serum on HIV-1 in vitro.Methods: The cytotoxicity of QKL was detected by MTT assay. H9/HIV-1_â…¢B cells labeled calcein-AM were treated with serially diluted QKL and its drug serum, and then co-cultured with MT-2 cells. After 2 hours, the cell fusion was observed with a fluoresencencemicroscope. The protective effect of QKL on cells infected by HIV-1 was detected by MTT. Viral replication was analyzed by measuring the level of p24 antigen in culture supernatants by p24 kit.Results: QKL was toxical to H9/HIV-1_â…¢B cells and MT-2 cells. The CC50 was 1/50.76 and 1/36.97 respectively. From cell fusion study, the EC₅₀ of QKL against early H9/HIV-1_â…¢B cells and MT-2 cells fusion was 1/235.29 with a selective index of 6.36. The EC₅₀ of QKL against the formation of syncytia of MT-2 induced by HIV-1_â…¢B was 1/198.02 with a SI of 5.36. By detecting the survival rate of co-culturing H9/HIV-1_â…¢B cells and MT-2 cells or MT-2 cell direct infected by HIV-1_â…¢B QKL was found to be protective to cells. The EC₅₀ was 1/166.67 and 1/144.93 with SI of 4.51 and 3.92 respectively. The EC₅₀ of QKL against P24 antigen production was 1/175.44 with SI of 4.75. The drug serum of QKL was also found to be effective to inhibit the cell fusion and protect cells infected by HIV-1_â…¢B.Conclusion: QKL and its drug serum have inhibitory effect on HIV-1 in vitro and it reacted through at least two clarified antiviral mechamisms. QKL inhibited not only entry into cell of HIV-1 but also the replication of HIV-1.