Screening Test And Pharmacology Study On Traditional Chinese Medicine Immunoenhancer Of Veterinary Field

The aim of the studies was to research and exploit the herb immunoenhancer with the ideal effect by a series of tests n vivo and vitro. Four parts were included in the studies. In part1, the immunosuppressive models were built, then the effective Formula of herb immunoenhancer was selected though in vivo tests; in part2, the acute toxicity and cytotoxicity of the herb immunoenhancer, which was selected from Part1, were tested; the in vitro immunological experiments were made in the ranges of safety concentration of the Formula in part3; the animal models were applied in part4, in which the function and mechanism of immune regulation of the Formula were validated from the aspect of immunology.Part 1 The selection of the effective herb immunoenhancer though in vivo testsThe mice were separated into two groups: the normal mice were in one group, and the immunosuppressive mice which were made by cyclophosphamide (Cy) were in the other group. They were all given Chinese herb medicine. In the applied four Formulas, Jade screen Powder (YPF) was the classical Formula, and the other three (TJJ1, TJJ2 and TJJ3) were designed according to the references. The YPF Formula was used as the control, the effects of TJJ1, TJJ2 and TJJ3 were estimated based on the indexes of Hematology, Weight, Organs and the Histological and Pathological changes of liver and spleen. Then the best Formula could be selected. The results demonstrated that: (1) the effects of TJJ1, TJJ2 and TJJ3 on the Hematological indexes (WBC,RBC,HGB,HCT,MCH,MCV)of normal mice were indistinctive (P>0.05), but the effects were distinctive to the immunosuppressive mice(P>0.05). The effect of TJJ2 was the best. All the Formulas could not make the lowed Hematological indexes back to the normal level. (2) The effects of all Formulas on the indexes of Weight and Organs of normal mice were indistinctive(P>0.05), but the effects were distinctive to the immunosuppressive mice(P<0.05). The Formulas of TJJ2 and YPF were the best two. (3) The Histological and Pathological splicing up showed that the splenocytes of the normal mice given by TJJ2 and YPF were clear and big, the hepatocytes were light degenerescence and necrosis. The results of immunosuppressive mice showed that the heavy degenerescence and necrosis of hepatocytes, but the white pulp was unclear. According to the above results, the effect of TJJ2 was best, and was selected to the Formula tested in the further experiments. The TJJ2 Formula was short for TJJ in the later studies.Part 2 The Acute Toxicity and Cytotoxicity of TJJThe in vivo and in vitro studies were made to determine the acute toxicity and cytotcoxicity of TJJ to the mice to make sure the safety of clinical application. In the in vivo studies, the Half Lethal Dose (LD50) and the Mean Tolerant Dose (MTD) were the indexes. The mice were given TJJ through intragastric administration at the dose of 40g/kg?w. The mice were still live after 1 week, then they were given the TJJ at the dose of 120g/kg?w everyday at the condition of the most tolerant dose. After 1 week, the mice were killed to observe the pathological change. In the vitro studies, the most safety concentration (TC0) of TJJ in CEF, L929 was determined through Observation and MTT. The results showed that: (1) the acute toxicity of TJJ was very low. The LD50 and MTD was above 40g/kg?w and 120g/kg?w, respectively. According to the toxicity level standard, the short period use of TJJ was safe. (2) The most safety concentration of TJJ in the CEF and L929 culture was 3.12g/L and 1.56g/L, respectively. The statistics made the basis for the later studies.Part 3 The effects of Herb immunoenhancer on the Function of Cellular Immune and anti-virus, anti-tumor Activity of the Mice through vivoThe immunosuppressive models were made by Cy, and the cells from the models and normal cells were cultured under the same conditions. The MTT was applied to measure the proliferation of the spleen lymphocytes and the activity of NK, and the key aspects of MTT, which were the cell concentration, the reaction time, and the different proportion, were optimized. The Neutral Red Absorbent Assay was applied to measure the function of Macrophage from the cavum abdominis. The above experiments were aimed to determine the effects of different concentration TJJ and YPF on the spleen lymphocyte transformation, the activity of NK, and the licking up function of Macrophage under the in vitro culture conditions. The results demonstrated that: (1)The spleen lymphocyte showed the exponent growth with the concentration of 1～6×109/L. With the proportion of 50:1, the reaction time of 18h, the NK could show the kill activity stably and high efficiently. (2)To the normal mice, the effects of TJJ and YPF of different concentrations on the spleen lymphocyte transformation and the activity of NK were indistinctive(P>0.05),but could enhance the activity of Macrophage. But for the immunosuppressive models, the effects of TJJ and YPF of concentrations between 100～1200μg/ml on the spleen lymphocyte transformation and the activity of NK were different(P>0.05), and the effect of TJJ was distinctive when the concentration was 400μg/ml(P<0.05).The virus infection titer was measured with Reed-Muench, the inhibition effect of TJJ and YPF on the proliferation of VSV was studied through L929-VSV detection system, and the induction activity of Chinese herb medicine on spleen lymphocyte to secrete IFN-γwas detected through mini cell pathogenic effect inhibition. The above experiments were aimed to determine if TJJ and VSV of different concentration could inhibit the proliferation of VSV, and induce the spleen lymphocyte to secrete IFN-γand its activity. The results showed that: (1) TJJ and YPF could not inhibit the proliferation of VSV in vitro. (2) The lymphocyte supernatant induced by TJJ and YPF demonstrated anti-VSV activity. With the concentration of 400μg/ml, TJJ showed the good result, and with the concentration of 500μg/ml, YPF showed the good result. TJJ and YPF of every concentration could not inhibit completely the CPE of L929 made by VSV(P>0.05).The above results demonstrated that TJJ and YPF had not the activity of anti-VSV in vitro, but the lymphocyte supernatant induced by them demonstrated anti-VSV activity to some degree.The NS-1 was cultured with different concentration of TJJ. The MTT was added after a certain time (12h,18h,24h,36h,48h),then the OD values were collected to determine the anti-tumor activity of the Formulas in vitro. The results showed that the Formulas could not inhibit the proliferation of tumor cell; with the extending of culture time ( over 24h), TJJ demonstrated the activity of promoting the growth of NS-1.Part 4 The effects of Chinese herb immunoenhancer on the immune function, anti-oxidation function and other indexes of the immunosuppressive mice models through in vivoThe immunosuppressive models were made by Cy. Then HC50 of mice serum was measured by spectrophotometry, the lymphocyte proliferation was detected by MTT, and the change of T cell subgroups was detected by flow cytometry. The above experiments were designed to make sure the effects of TJJ on the immune function of normal mice and immunosuppressive mice. The activity of IL-2 was measured through the proliferation of thymus cell, the activity of IFN-γwas measured through mini CPE inhibition. The aim of the above two experiments was to demonstrate the effects of herb Formulas on the secretion of cytokines from lymphocytes of immunosuppressive mice. The content of NO, MDA and SOD in the serum was measured to determine the effects of TJJ and YPF on the anti-oxidation activity of normal mice and immunosuppressive mice. The results showed that: (1) After being inoculated SRBC, the secretion of hemolysin of immunosuppressive mice was inhibited, and the decrease was distinctive((P<0.05). The value of HC50 in the models of being given the TJJ and YPF were higher than the group of not being the Formulas, and the discrepancy was distinctive(P<0.05). The effect of TJJ was better than the YPF, and the discrepancy was distinctive(P<0.05). (2) The lymphocyte transformation rate within 48h of the tested groups was almost same as that of the lymphocyte from the control group, in which the pastille serum was added into the lymphocyte culture. The results meant the TJJ could enhance clearly the lymphocyte transformation for the mice with abnormal immunity. The lymphocyte transformation rate of TJJ group was higher than that of control group, Cy group and YPF group(P <0.05).TJJ and YPF Formula could enhance the level of CD3 and CD8 of the immunosuppressive mice, and antagonize the increase of the CD4/CD8 ratio caused by Cy. And there was distinctive statistics discrepancy(P<0.05). (3) The optimal cell concentration of lymphocyte proliferation in thymus was 3～8×109/L. The supernatant from spleen lymphocyte of tested groups showed strong IL-2 activity when the dilution degree of supernatant was between 1:4～1:32, and the discrepancy was distinctive. The activity of IFN-γof Cy+ TJJ group was highest(8.1414±0.5589)㏒ 2, and the discrepancy was distinctive compared with the models(P <0.05).(4) The TJJ and YPF could lower clearly the content of NO, MDA and increase the content of SOD in the serum of the normal mice and immunosuppressive mic(e P<0.05),which meant the TJJ had the effect of anti-oxidation and immune regulation.Conclusion(1) The TJJ Formula was mini toxic and safe in application. The acute toxicity LD50 and MTD was above 40g/kg?w and 120g/kg?w, respectively. According to the toxicity level standard, the short period use of TJJ was safe. The most safety concentration of TJJ in the CEF and L929 culture was 3.12g/L and 1.56g/L, respectively.(2) According to all results that we had tested, we considered TJJ had the effect of anti-oxidation and immune regulation significantly, especially to organism with abnormality function.(3) TJJ is better than YPF.