| PurposeChoosing QIANG JI JIAN LI FANG (QJJLF) as the research object, copying animal spleen deficiency models and cultivating the cell of thymus and spleen in rats of experimental spleen deficiency in vitro. Through quantitative analysis method of pharmacology and immunology to further explore the mechanisms of QJJLF in preventing and curing the spleen deficiency, and provide the basis for the next research and clinical application.Method1. TheoryThe theory and experiment research of the components in QJJLF, spleen deficiency, serum pharmacology of Chinese traditional medicine are reviewed in this paper. On the basis of the previous studies, taking medicinal serum of QJJLF as the breakthrough point to carry out experiments by high sensitivity and specificity indexs, such as TNF-α, IL-2, IL-6, IFN-γ, thyroid hormones and amylase.2. Experimental studyEach rat was infused QJJLF (dosage:23.4g/kg/day) into stomach until steady serum level was monitored and got the medicinal serum of QJJLF. The animal model of spleen deficiency was built by Dahuang and the parameters T3,T4,AMS were detected. Then the cells of thymus and spleen were separately analyzed. The cells were divided into five groups:three experimental groups (low dosage, middle dosage and high dosage), two control groups:normal serum group, fetal bovine serum(FBS) group. ConA was added to incubate the cells for 72 hours. Then the supernatant was collected to detect the cell factor such as TNF-α, IL-2, IL-6, IFN-γ. The rest cells were applied to determine the protein expression of Fas and PCNA by immunohistoc- hemistry. The mechanisms of QJJLF in preventing and curing the spleen deficiency and its effect on immune function were revealed by the efficacy of the treatment group changes. Providing the basis for the next research and clinical application.Result(1). Generally symptoms:The animal model of spleen deficiency began to weight loss, narrowed eyes, lazy, gathers, timidity, then strip body hair loose stools, waiting for a symptom, and with the extension of time symptoms gradually increase after applied Dahuang three days. The various manifestations stabilized 11 days later. It is similar to the clinical manifestations of patients with spleen deficiency. Compared to the normal control group, weight and hypothermy were decreased significantly in spleen deficiency model rats 20 days later (P<0.01,0.05).(2). Effect on T3,T4,AMS after succueeful modeling by Dahuang:Compared to the normal control group, T3 and AMS were decreased significantly in spleen deficiency model rats after succueeful modeling (P<0.01). The content of T4 was decreased slightly but didn't have significant difference.(3). Effect on TNF-α:For the thymus cell, compared to the FBS control group, TNF-αwas increased significantly in the groups with low dosage, middle dosage and high dosage of QJJLF(P<0.01). When compared to the normal serum group, TNF-αalso increased in the three groups (P<0.01,0.05). And the groups with middle dosage and high dosage of QJJLF increased significantly (P<0.01).For the spleen cell, compared to the FBS control group, TNF-αwas increased significantly in the groups with low dosage, middle dosage and high dosage of QJJLF(P<0.01). Compared to the normal serum group, TNF-αincreased significantly in the high dosage groups(P<0.01). While the low dosage and middle dosage didn't have significant difference. Compared to the low dosage group, TNF-αwas increased significantly in the groups with middle dosage of QJJLF(P<0.01). It shows that QJJLF can stimulate cells of thymus and spleen to secrete TNF-α.(4). Effect on IL-2:For the thymus cell, compared to the FBS control group and the normal serum group, IL-2 was increased significantly in the groups with low dosage, middle dosage and high dosage of QJJLF(P<0.01).For the spleen cell, compared to the FBS control group and the normal serum group, IL-2 was increased significantly in the groups with low dosage and high dosage of QJJLF(P<0.01). It shows that QJJLF can stimulate cells of thymus and spleen to secrete IL-2.(5). Effect on IL-6:For the thymus cell, compared to the FBS control group, IL-6 was increased significantly in the groups with low dosage, middle dosage and high dosage of QJJLF(P<0.01). Compared to the normal serum group, those three groups can stimulate spleen cell to release IL-6(P<0.01-0.05), the most significant effect was got in the middle dosage group(P<0.01).For the spleen cell, compared to the FBS control group, IL-6 was increased in the groups with low dosage, middle dosage and high dosage of QJJLF(P<0.05). While compared to the normal serum group, those there groups didn't have significant difference. It shows that QJJLF can stimulate cells of thymus and spleen to secrete IL-6.(6). Effect on IFN-γ:Compared to the normal serum group, IFN-γin the supernatant was increased significantly in the groups with low dosage, middle dosage and high dosage of QJJLF(P<0.01). But IFN-γtrend to decrease with the increase of the proportion of drug-containing serum.(7). Effect on the expression of thymus PCNA:Thymus PCNA in the middle dosage of QJJLF was up-regulated. And the positive area of expressed cells, positive area ratio and integrated optical density in the middle dosage group were increased significantly when compared to the FBS control group(P<0.01). Compared to the low dosage and high dosage of QJJLF, the expression of thymus PCNA in the middle dosage was up-regulated significantly(P<0.01).(8). Effect on the expression of thymus Fas:Thymus Fas in the middle dosage of QJJLF was down-regulated. And the positive area of expressed cells, positive area ratio and integrated optical density in the middle dosage group were decreased significantly when compared to the FBS control group (P<0.01). Compared to the low dosage and high dosage of QJJLF, the expression of thymus Fas in the middle doseage was down-regulated significantly(P<0.01). ConclusionQJJLF is an effective drug in preventing and curing the spleen deficiency. It is proved in this paper that QJJLF can stimulate cells to secrete some cytokines of celluar immune function such as TNF-α,IL-2,IL-6,IFN-γ. IL-2 and IL-6 can stimulate T and B cell to secrete immune antibody. TFN-αand IFN-γare secreted by lymphocyte. At the same time the expression of PCNA is up-regulated and the thymus nuclear proliferation. Those increase reflect the activation of host immune system. These cytokines interactive to improve the body's immune system. So that the original disturbance of cytokines return to normal physiological. This is so called "balance between yin and yang" in the Chinese Medicine. And the down-regulated expression of Fas proves the cells proliferation from the opposite. It proves that QJJLF can adjust the disordered immune system when spleen deficiency occurred. Especially making the immune organs and immune cells revitalization. At the same time, cellular immunity and humoral immunity promote each other, so that the body reaches a new immune balance.All indexes show that QJJLF has effects on the regulation of the immune parameters. So we could speculate that the mechanism it preventing and curing the spleen deficiency is through a way of cellular and humoral immune responses. At the same time we found that the best dosage of blood concentration is 20%, which can further guide the dosage of oral drug and injection in clinical practice.
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