| Objective:1.To observe the therapeutic effect of Jian Pi Zi Shen Xie Huo recipe and its dissolving recipe on ITP mouse model based on "spleen and kidney airfire rela ted pathogenesis".2.To investigate the action of jianpi zishen xiehuo recipe and its dissection on ITP mice.3.Clarify the mechanism of action between Breg and Jian Pi Zi Shen Xie H uo recipe and the scientific connotation of ITP "spleen and kidney qi and fire r elated" pathogenesis.Methods:1.The model was established using the modified passive immune induction method of anti-cd41 monoclonal antibody.ITP animal models were induced b y intraperitoneal injection of 200 ul 68ug/kg at day 1,200 ul 102ug/kg at day 2,and 200 ul 136ug/kg of anti-mouse CD41 monoclonal antibody(MWReg30)at day 3 to day 13 in Balb/c mice.2.Experimental grouping and treatment intervention:(1)blank control grou p: intraperitoneal injection with PBS and gavage with normal saline;(2)model control group: intraperitoneal injection of anti-cd41 antibody + gavage with no rmal saline;(3)prednisone control group: anti-cd41 antibody intraperitoneal inje ction + prednisone gavage treatment;(4)TCM treatment group:(1)jianpi yiqi group: anti-cd41 antibody intraperitoneal injection + jianpi yiqi treatment;(2)n ourishing kidney-yin group: anti-cd41 antibody intraperitoneal injection + kidne y-nourishing kidney-yin treatment;(3)heat-clearing and fire-purging group: anti-cd41 antibody intraperitoneal injection + heat-clearing and fire-purging prescrip tion treatment;(4)jianpi zishen xie huo group: anti CD41 antibody intraperiton eal injection + jianpi zishen xie huo fang treatment.Continuous intragastric ad ministration for 10 days(the maximum duration of administration required by t he experiment).3.During the modeling and administration,the general state,body weight a nd skin and mucosal bleeding of mice were observed and recorded dynamicall y;At the same time,on day 0,day 3 of the model establishment and day 10 of the drug intervention(at the end of the administration cycle),the whole blood of 20 ul was taken from the tail vein of mice for platelet count detection and analysis.After the successful establishment of the model,the total number of megakaryocytes and the number of megakaryocytes producing plates in the femoral bone marrow of mice in each group were observed and recorded at r andom,and the model evaluation was conducted,as well as the efficacy analy sis of the animal model of invigorating spleen,nourishing kidney,relieving fire prescription and its dissection for ITP treatment.4.On the day of the end of administration,the mice were sacrificed by ce rvical dislocation method,and their spleenas were taken aseptically.200 mesh sterile filter was ground,and the red blood cell lysate was cracked to produce the suspension of splenic mononuclear cells.The recognized mouse regulatory B cell(Breg)phenotype CD1 dhi CD19+CD5+B cell subsets were selected as t he observation objects,and the flow cytometry analyzer was used to analyze t he proportion of CD1 dhi CD19+CD5+ Breg cells in spleen tissues after treatme nt in blank control group,model control group,prednisone control group and e ach treatment group of traditional Chinese medicine.After the regulatory B cel ls(Breg)were isolated by magnetic bead sorting technology,real-time PCR wa s used to analyze the expression level of il-10 mrna in Breg cells between eac h group.After sorting the Breg and excluding Breg spleen organization monon uclear cell system respectively to join CD3 / CD28 functional antibody beads,trained 72 hours after culture supernatant,ELISA method for each group of sp leen tissue Breg cells in the mononuclear cell system under the condition of e xistence and there is no related to inflammatory factor in the culture supernata nt IFN-gamma,the amount of TNF alpha test.Results:1.Body weight comparison: the body weight of mice in the blank control group showed that the body weight comparison before and after the modeling was not statistically significant(Z=-1.918,P=0.055>0.05),and there was no dif ference in body weight before and after the modeling.The body weight of mi ce in each group induced by the model was as follows: body weight before a nd after modeling,t=4.097,P=0.0001<0.001,which was statistically significant.Body weight after modeling was lower than that before modeling.Comparison of platelet count: there was no difference between the blank control group an d the blank control group before and after modeling(Z=-0.757,P=0.449 > 0.05).Model induced platelet counts before and after each building(t = 66.871,P = 0.0001)there were significant differences,significantly lower platelet count after building(3)the general state of comparison: model induced by each gro up generally worse than normal control group,main show is poorer mental stat e and decreased activity decreases,hair messy,diet,skin and mucosa hemorrha ge is rare and difficult to take the initiative to absorb,etc;No significant subc utaneous hemorrhage was observed in the normal control group.(4)megakaryo cytes and placental megakaryocytes: compared with the normal control group,t he model induced the total number of megakaryocytes in the bone marrow of mice in each group increased,and the number of placental megakaryocytes dec reased.2.Body weight comparison:(1)body weight was increased in the blank co ntrol group(P=0.0001),spleen nourishing qi group(P=0.0001),spleen nourishin g kidney nourishing fire group(P=0.0001)and prednisone group(P=0.027)co mpared with the model control group,and the difference was statistically signif icant(P < 0.05).There was no significant difference between the model contro l group and the nourishing kidney-yin group(P=0.443)and the heat-clearing a nd fire-purging group(P=0.999)(P>0.05).Comparison of platelet count: the pl atelet count of the blank control group(1046 75.01)was higher than that of t he other groups(P<0.001).The platelet count of the model control group(54975.49)was lower than that of the heat-clearing and heat-purging group(71354.78)(P<0.001),the spleen-nourishing and kidney-purging group(735 54.78)(P<0.001)and the prednisone group(783 49.00)(P<0.001).There was no signi ficant difference between the two groups(P>0.05).Comparison of each treatme nt group: the platelet count in the prednisone group was higher than that in th e spleen-nourishing kidney-purging fire group and heat-clearing and fire-purging group(P < 0.05),and there was no statistical difference in the platelet count between the spleen-nourishing kidney-purging fire group and heat-clearing and fire-purging group(P>0.05).(3)general comparison: the mice in the model c ontrol group and nourishing kidney-yin group showed poor mental state,slow r eaction,less diet,skin mucosa bleeding and long active healing time,etc.;Co mpared with the model control group,the spleen-invigorating qi group showed better mental state,thicker hair,slightly improved skin and mucosal bleeding,a nd more smooth intraperitoneal injection.In the heat-clearing and fire-purging group,the mental state was general,and there were more cases of cool urine and turbid anus,subcutaneous hemorrhage was relatively fixed,and new hemor rhage sites were rare.The mice in the spleen-strengthening,kidney-nourishing and fire-purging group showed less new blood loss at the injection site and be tter mental state than the model control group.In the prednisone group,the ac tivity was increased,the reaction was sensitive,the diet was increased a little,there were bilateral underarm abscesses and chest and abdomen abscesses occas ionally,subcutaneous new hemorrhage was rare.3.The proportion of Breg cells in the spleen tissues of each group after a dministration:(1)the Berg/B value of the blank control group(1.35 0.33)was higher than that of the model control group(0.60 0.22),P=0.014,and the dif ference was statistically significant(P < 0.05).The Berg/B value of the model control group was lower than that of the prednisone group(2.13 0.36),and t he difference of P=0.0001 was statistically significant(P < 0.001).The differen ce of P=0.025 was statistically significant(P < 0.05).The difference was statis tically significant(P < 0.05).It was lower than the nourishing kidney-yin grou p(1.42 0.27),P=0.003,and the difference was statistically significant(P < 0.001).There was no difference in Berg/B between the two groups(P > 0.05).C omparison of each treatment group: there was no difference in Berg/B value b etween the prednisone group and the spleen-nourishing kidney-nourishing fire-p urging group(both P > 0.05),and there was a statistically significant differenc e between the two groups in P=0.044(P < 0.05).There was no difference in Berg/B values between the three groups(P > 0.05).4.The expression of il-10 mrna in Breg cells of mice in each group after administration:(1)the expression of il-10 mrna in the blank control group(1.22 0.18)was higher than that in the model control group(0.48 0.06),P=0.001,and the difference was statistically significant(P < 0.05).(2)the Breg expres sion level of il-10 mrna in the model control group was lower than that in th e spleen-strengthening,kidney-nourishing and fire-purging group(1.34 0.38),P=0.002,and the difference was statistically significant(P < 0.05).The difference was statistically significant(P < 0.05).The difference was statistically signific ant(P < 0.05).(3)comparison of each treatment group: the Breg expression o f il-10 mrna in the spleen-strengthening,kidney-nourishing and fire-purging gro up was higher than that in the prednisone group,P=0.042,and the difference was statistically significant(P < 0.05).The difference was statistically significa nt(P < 0.05).Compared with prednisone group,P=0.629(P > 0.05),it can be considered that there is no difference in the value of il-10 mrna expression in Breg between the two groups5.Each Breg cells in mice after the treatment of inflammatory factors asso ciated IFN-gamma:(1)the effect of each treatment group Breg cells of sple en tissues contained in a single nuclear cell system culture supernatant of IFN-gamma production comparison: the production of IFN-gamma in blank co ntrol group(3.60 + 0.87)is lower than the model control group(7.01 + /-0.38),with significant difference(P = 0.0001 statistical significance(P < 0.001).(2)the secretion of IFN-in the model control group was higher than that in the heat-clearing and heat-purging group(4.04 1.52)(P=0.001),the prednisone group(3.81 0.22)(P=0.0001),and the spleen-nourishing and kidney-purging gro up(3.19 0.31)(P=0.0001),with P < 0.001 difference.(3)comparison of each treatment group: there were P > 0.05 between the spleen nourishing kidney rel ieving fire group,clearing heat relieving fire group and prednisone group,sugg esting that there was no difference in the secretion of IFN-in the supernatant culture between the three groups.(2)comparison of IFN-secretion in the supe rnatant of Breg cell culture system and Breg cell culture system in each group:(1)including and excluding Breg group meet in blank control group of norm al distribution and variance qi(F = 3.653,P = 0.129),between the two group s using independent sample t-test,the results suggest t = 5.487,P = 0.005,the difference was statistically significant(P < 0.05),can be thought of in blank control group including and excluding Breg system within the culture supernata nt of IFN-gamma production vary,and not expressed in the Breg system co ntent is higher.(2)in the model control group,the breg-containing and breg-fr ee groups met the normal distribution and homogeneity of variance(F=0.113,P=0.754).Independent sample t test was used between the two groups,and the results indicated that t=-2.392,P=0.075.There was no difference in IFN-secr etion in the supernatant cultured in the breg-containing and breg-free systems b etween the two groups(P > 0.05).Group(3)heat purging fire including and excluding Breg group meet normal distribution and variance qi(F = 4.354,P=0.105),between the two groups using independent sample t-test,the results sug gest t = 2.790,P = 0.049,the difference was statistically significant(P < 0.05),a group can be thought of as heat purging fire including and excluding Br eg system within the culture supernatant of IFN-gamma production vary,and not expressed in the Breg system content is higher.(4)spleen zi kidney purging fire,including and excluding Breg group meet normal distribution and vari ance qi(F = 1.138,P = 0.346),between the two groups using independent sa mple t-test,the results suggest t = 3.477,P = 0.025 difference was statistically significant(P < 0.05),can be thought of spleen zi kidney purging fire group including and excluding Breg system within the culture supernatant of IFN-g amma production vary,and not expressed in the Breg system content is higher.5.Prednisone group,including and excluding Breg satisfy normal distribution and variance qi(F = 2.381,P = 0.198),between the two groups using indepe ndent sample t-test,the results suggest t = 8.947,P = 0.001,the difference wa s statistically significant(P < 0.05),can be thought of in prednisone group inc luding and excluding Breg system culture supernatant of IFN-gamma product ion vary,and not expressed in the Breg system content is higher.6.Effects of Breg cells on TNF-in mice of each group after administratio n:(1)comparison of TNF-secretion in splenic mononuclear cell culture supern atant containing Breg cells in each treatment group:(1)comparison of TNF-s ecretion between blank control group and model control group P=0.078,no dif ference was found in TNF-secretion between the two groups(P>0.05).(2)co mparison of model control group with nourishing kidney-yin group and clearin g away heat and relieving fire group showed P<0.001,and the difference was statistically significant.There was no significant difference in TNF-secretion b etween the two groups(P>0.05).(2)each group contains Breg spleen organizat ion system of mononuclear cells and mononuclear cells do not contain Breg ce lls system interferons alpha production comparison:(1)including and excluding Breg group meet in blank control group of normal distribution and variance q i(F = 0.001,P = 0.974),between the two groups using independent sample ttest,the results suggest t = 3.44,P = 0.026 statistically difference(P < 0.05),can be thought of in blank control group including and excluding Breg system within the culture supernatant of interferons alpha production vary,and not ex pressed in the Breg system content is higher.(2)in the model control group i ncluding and excluding Breg group meet normal distribution and variance qi(F= 1.357,P = 0.309),between the two groups using independent sample t-test,the results suggest t = 5.901,P = 0.004 statistically difference(P < 0.05),ca n be thought of in the model control group including and excluding Breg syst em culture supernatant of interferons alpha production vary,and not expressed in the Breg system content is higher.(3)the spleen invigorating qi group with and without Breg met the normal distribution and homogeneity of variance(F=2.1,P=0.221).Independent sample t test was used between the two groups,a nd the results showed that t=-2.13,P=0.1 difference was not statistically signifi cant(P > 0.05).It could be considered that there was no difference in the sec retion of IFN-in the supernatant cultured in the spleen invigorating qi group with and without Breg system.(4)the nourishing kidney Yin group including and excluding Breg group meet normal distribution and variance qi(F = 1.359,P =0.308),between the two groups using independent sample t-test,the result s suggest t = 4.985,P = 0.008,with statistical significance(P < 0.05),can be thought of in nourishing kidney Yin group including and excluding Breg syst em within the culture supernatant of interferons alpha production vary,and not expressed in the Breg system content is higher.Group(5)heat purging fire i ncluding and excluding Breg group meet normal distribution and variance qi(F= 0.006,P = 0.942),between the two groups using independent sample t-test,the results suggest t = 20.793,P = 0.0001,with significant difference statistic ally significant(P < 0.001),a group can be thought of as heat purging fire in cluding and excluding Breg system within the culture supernatant of interferons alpha production vary,and not expressed in the Breg system content is higher.6 spleen zi kidney purging fire,including and excluding Breg group meet nor mal distribution and variance qi(F = 4.858,P = 0.92),between the two group s using independent sample t-test,the results suggest t = 2.925,P = 0.043,wi th significant difference statistically significant(P < 0.05),can be thought of s pleen zi kidney purging fire group including and excluding Breg system within the culture supernatant of interferons alpha production vary,and not expressedi n the Breg system content is higher.All landowners in prednisone group inclu ding and excluding Breg group meet normal distribution and variance qi(F =0.879,P = 0.401),between the two groups using independent sample t-test,the results suggest t = 5.275,P = 0.006,with significant difference statistically sig nificant(P < 0.05),can be thought of in prednisone group including and exclu ding Breg system culture supernatant of interferons alpha production vary,and not expressed in the Breg system content is higher.Conclusion:1.Jianpi zishen xiehuo recipe,qingrezhihuo recipe and prednisone all have effects on improving the platelet count in ITP model,and jianpi zishen xiehuo recipe has the best effects on improving the symptoms,preventing bleeding a nd improving the platelet count in ITP model.2.Invigorating the spleen,nourishing the kidney and relieving the fire pres cription can inhibit the secretion of related inflammatory factors by Th1 cells a nd adjust the balance of Th1/Th2 by increasing the expression of Breg and im proving its immunomodulatory function,thus playing a therapeutic role in the I TP model.3.There is a certain material basis for ITP "spleen and kidney airfire relat ed pathogenesis" theory,which is speculated to be related to Breg quantity abn ormality and dysfunction. |
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