Lizhong Wan And Four Gentlemen Tang Spleen Deficiency Syndrome Square Cards Relevance Of Experimental Research

As a bridge between theory and treatment of Traditional Chinese Medicine, formula and syndrome is important in the study on Traditional Chinese Medicine. Effects of theory of Traditional Chinese Medicine on treatment and composing formula can be understood deeply by works on correlation between formula and syndrome. Moreover, biological data can be provided on Chinese herbs usage based on TCM theory. Present studies on TCM were almost focused on effects of formula on syndrome or diseases, correlation between formula and syndrome is seldom committed. In this experiment, correlation was emphasized. ObjectivesTo better understand the correlation between formula and syndrome, reserpine induced spleen deficiency was used, effects of Lizhong Pill and Sijunzi formula on spleen deficiency were investigated on observation on rats, basic activity, clotting time, digest related function, enteric mucosal immune, and enteric neuro-endocrine functions. Method of cluster analysis may provide insights into correlation between Lizhong Pill & Sijunzi formula and spleen syndrome. Materials and MethodsSD rats were injected with reserpine every day for two weeks. Appearance of rats were observed, weights measured every week, basic activity measured with patented electronic equipment and analyzed with collection & analysis system of physiological signal, clotting time measured, serum from SD rats stored -20 degree, counts of Peyer's patch in intestinal, intestinal fixed in 4% formaldehyde and stored in liquid nitrogen for paraffin section and frozen section respectively, pathological and ultrastructural changes measured with methods of HE staining and scanning electron microscope, cytokines of IL-1, IL-6, IFN- γ and TNF-α , gastrin in serum, vasoactive intestinal pepude (VIP) and substance P (SP) in jejunum detected with methods of enzyme linked immunosorbent assay (ELISA) and radio-immunity, expression of TNF- α and TGF- β in jejunum with method of immunohistochemistrystaining, subtype of T lymphocyte in blood and enteric Peyer's patch with flow cytometer, activity of spleen lymphocyte proliferation activated by ConA and LPS with method of MTT reduction assay (MTT). Results1 General observation:Rats injected with reserpine for two weeks were found with including cachexia, lassitude, poor appetite, reduction of body weight, low-grade depilation and diarrhea, but not death, which is as same as spleen deficiency syndrome of Traditional Chinese Medicine. After treatment with Lizhong Pill and Sijunzi formula in two level of dose separately for two weeks, above symptoms of rats were alleviated in different degree in different group.2 Basic activityBasic activity of spleen deficiency syndrome rats was lessen than mat of normal rats, and significantly increased by treatment with Lizhong Pill and Sijunzi formula.3 Clotting timeThere was no significant difference between normal, model and treatment groups.4 Counts of Peyer' s patchThere was no significant difference between normal, model and treatment groups.5 Pathological alterations of jejunumNormal Control: intestinal villi structure was normal. There were not hyperaemia, hydropsia, necrosis and inflammatory cell infiltration. Enteric cavity was clean without exfoliation. Organization of villi cells appeared normal.Spleen deficiency syndrome rats: alterations included a villi necrosis with infiltration of polymorphonuclear and mononuclear cells, connective tissue hydropsia, enteric cavity was fall of villi necrosis.Treated with Lizhong Pill and Sijunzi formula: spleen deficiency syndromes appeared in deficiency-without-treatment animals intestinal were reduced significantly.6 Ultrastructure alterations of jejunumNormal Control: digital villi with clean surface projected into enteric cavity orderly,villi interval was clear.Spleen deficiency syndrome rats: villi necrosis with dirty surface can be observed, enteric cavity Ml of villi necrosis disappeared..Treated with Lizhong Pill and Sijunzi formula: spleen deficiency syndromes appeared in deficiency-without-treatment animals intestinal were reduced significantly.7 Detection of IL-1, IL-6, IFN- y, TNF- a and gastrin in serum:Reserpine increased the level of cytokine IL-6 in serum in vivo, together with gastrin, which were reversed by treatment with Lizhong Pill and Sijunzi formula similarly, whereas IL-1, IFN- Y and TNF- a , which was down-regulated in spleen deficiency syndrome animals, was back to normal in response to treatment.8 subtype of CD4+ and CD8+ T cells and ratio of CD4+/CD8+ in bloodRatio of CD4+/CD8+ in blood of spleen deficiency rats were antagonized significantly by treatment with Lizhong Pill and Sijunzi formula similarly, but there were little effects on increment of CD8+ and decrement of CD4+T cells.9 Subtype of CD4+ and CD8+ T cells and ratio of CD4+/CD8+ in intestinal PP nodesThere was no significant difference between spleen deficiency and normal rats, and Lizhong Pill and Sijunzi formula have no significant effect on subtype of lymphocytes in intestinal PP nodes.10 Expression of TNF- a and TGF- P in jejunumLizhong Pill and Sijunzi formula reversed the increase of TNF- a and decrease of TGF- P in jejunum of spleen deficiency rats to normal level significantly.11 Detection of VIP and SP in ileumReserpine increased the level of SP and decreased VIP in ileum in vivo, which were reversed by treatment with Lizhong Pill and Sijunzi formula similarly.12 Spleen lymphocyte ProliferationIn spleen deficiency syndrome rats, lymphocyte proliferation activated by ConA was down-regulated, but not by LPS. Proliferation by ConA can only be increased by treatment of Lizhong Pill in low dose and Sijunzi formula in high dose.Conclusions? Lizhong Pill and Sijunzi formula can alleviate symptoms of reserpine induced spleen deficiency rats;? Lizhong Pill and Sijunzi formula have no effect on clotting time, counts of PP nodes and subtype of CD4, CD8 T lymphocyte and ratio of CD4/CD8 in enteric PP nodes of reserpine induced spleen deficiency rats;? Lizhong Pill and Sijunzi formula can improve enteric pathological and ultrastructure alternation together with decrease of serum gastrin, to regulate ingest function, of reserpine induced spleen deficiency rats;? Lizhong Pill and Sijunzi formula can regulate neuroendocrine net in reserpine induced spleen deficiency rats by up-regulate of VIP and down-regulate ofSP.;? Lizhong Pill and Sijunzi formula can regulate general immune function of reserpine induced spleen deficiency rats by the ways of spleen T lymphocyte proliferation, serum level of IFN- Y, TNF- a , IL-1, and IL—6, ratio of CD4+/CD8+T lymphocyte in blood;? Lizhong Pill and Sijunzi formula can increase expression of TGF- P and reduce TNF— a in jejunum in different degree.? Lizhong Pill and Sijunzi formula correlated differently with reserpine induced spleen deficiency syndrome.