| Purpose: Modified Sijunzi decoction was used to intervene ITP model mice and observe its effect on platelets of ITP mice.The contents of IL-6,IL-17,IL-22,IL-10 and TGF-β in serum of each group of mice were detected to explore the improvement effect of modified Sijunzi Decoction on Inflammatory state of ITP mice.By detecting the ratio of Th17/Treg cells,this paper expounds the important role of Th17/Treg imbalance in the pathogenesis of ITP and the improvement effect of Modified Sijunzi Decoction on Th17/Treg balance.By detecting the expression level of RORγt and Foxp3 m RNA and the activation state of Jak/Stat3 signal pathway,it is clarified that Modified Decoction regulates the expression of RORγt,Foxp3 m RNA and protein by inhibiting the activation of Jak/Stat3 signal pathway,so as to realize the role of regulating Th17/Treg balance.Materials and methods: Platelet rich plasma was prepared from 40 mice and mixed with Freund’s complete adjuvant to prepare allergens,which were injected subcutaneously into the back,groin and paw of 10 guinea pigs.Three times of enhanced sensitization were performed on Day7,day14 and day28.The method was the same as the initial sensitization.The antigen solution was platelets suspended in Freund’s incomplete adjuvant.On day35 the whole blood of guinea pig artery was collected by abdominal aortic puncture,then the supernatant was collected and mixed to prepare GP-APS.On day29 purchased the second batch of 60 mice.After one week of adaptive feeding,on day36 randomly divided the mice into two groups: blank group(10 mice)and model group(50 mice).GP-APS was injected into the model mice by intraperitoneal injection at a dose of 100 u L/20 g body weight.The mice in the blank group were fed normally without treatment.On day43 observed the formation of subcutaneous ecchymosis in mice,detected the content of platelets and evaluated the model.After successful modeling,the model mice were randomly divided into model group,highdose group,medium dose group,low-dose group and prednisone group.The scheme is as follows: blank group: without treatment,continue to feed normally for 10 days;Model group:oral gavage of normal saline,0.17ml/10 g body weight,once a day,for 10 days;High,medium and low dose groups: oral gavage of high,medium and low concentration of traditional Chinese medicine solution,0.17ml/10 g body weight,once a day for 10 days;Prednisone group: oral administration of 0.15mg/ml prednisone suspension,0.17ml/10 g body weight,once a day for 10 days.2 hours after the last administration,peripheral blood was collected and blood routine was tested;The contents of TPO,IL-6,GPⅡB/Ⅲa,IL-17,IL-22,IL-10 and TGF-β in plasma were detected by ELISA;Th17/Treg cell ratio was detected by flow cytometry;The expressions of RORγt and Foxp3 m RNA in PBMC were detected by RT-q PCR;The expression levels of p-jak1/2/3,p-Stat3 and RORγt in PBMC were detected by WB and ICC.Results:1.Model evaluation results showed that: Compared with blank control group,the peripheral blood platelet count of mice in the modeling group was significantly decreased(P<0.01),and the mean platelet volume was significantly increased(P<0.01),with statistically significant differences.2.The results of routine blood test showed that: Compared with the blank group,the platelet count in peripheral blood of mice in other groups decreased significantly(P<0.01),and the average platelet volume increased significantly(P<0.01),with statistically significant differences;Compared with the model group,the platelet count in peripheral blood of mice in each intervention group increased significantly(P<0.01),and the average platelet volume decreased significantly(P<0.01),with statistically significant differences;Compared with the high dose group,the platelet count in the peripheral blood of mice in the medium dose group,low dose group and prednisone group decreased in varying degrees(P<0.05),and the average platelet volume increased in varying degrees(P<0.05),with statistically significant differences;Compared with the middle dose group,the platelet count in peripheral blood of mice in the low dose group decreased significantly(P<0.01),and the average platelet volume increased significantly(P<0.05),with statistically significant differences.3.The results of plasma TPO,IL-6 and GPⅡb/Ⅲa showed that: Compared with the blank group,the contents of TPO and GPⅡb/Ⅲa in plasma of mice in other groups decreased significantly(P<0.01),and the contents of plasma IL-6 in model group,medium dose group,low dose group and prednisone group increased significantly(P<0.01),with statistically significant differences;Compared with the model group,the content of TPO in plasma of mice in each intervention group increased in varying degrees(P<0.05),the content of IL-6decreased significantly(P<0.01),and the content of GPⅡb/Ⅲa in plasma of mice in high dose group,medium dose group and prednisone group increased significantly(P<0.01),with statistically significant differences;Compared with the high dose group,the content of plasma TPO in the medium dose group,low dose group and prednisone group decreased significantly(P<0.01),the content of GPⅡb/Ⅲa decreased in varying degrees(P<0.05),and the content of IL-6 increased significantly(P<0.01),with statistically significant differences;Compared with the middle dose group,the contents of plasma TPO and GPⅡb/Ⅲa in the low dose group decreased significantly(P<0.01),and the content of IL-6 increased significantly(P<0.01),with statistically significant differences.4.Plasma levels of IL-17,IL-22,IL-10 and TGF-β showed that: Compared with the blank group,the content of IL-10 and TGF-β in plasma of mice in other groups decreased significantly(P<0.01),The levels of IL-17 and IL-22 in plasma of model group,medium dose group,low dose group and prednisone group were significantly increased(P<0.01),with statistically significant difference;Compared with the model group,the contents of IL-17 and IL-22 in plasma of mice in each dose group of traditional Chinese medicine and prednisone group decreased significantly(P<0.01),and the contents of IL-10 and TGF-β in plasma of mice in high dose group,medium dose group and prednisone group increased significantly(P<0.01),with statistically significant difference;Compared with the high dose group,the contents of IL-17 and IL-22 in plasma of mice in medium dose group,low dose group and prednisone group increased significantly(P<0.01),and the contents of IL-10 and TGF-βdecreased significantly(P<0.01),with statistically significant difference;Compared with the middle dose group,the contents of IL-17 and IL-22 in plasma of mice in the low dose group increased significantly(P<0.01),and the contents of IL-10 and TGF-β decreased significantly(P<0.01),with statistically significant difference.5.The detection results of Th17/Treg cell ratio in mouse PBMC showed that: Compared with the blank group,the percentage of Th17 positive cells in PBMC of other groups increased significantly(P<0.01),and the percentage of Treg positive cells decreased significantly(P<0.01),with statistically significant differences;Compared with the model group,the percentage of Th17 positive cells in PBMC of mice in each dose of traditional Chinese medicine group and prednisone group decreased significantly(P<0.01),and the percentage of Treg positive cells increased significantly(P<0.01),with statistically significant differences;Compared with the high dose group,the percentage of Th17 positive cells in PBMC of mice in medium dose group,low dose group and prednisone group increased significantly(P<0.01),and the percentage of Treg positive cells decreased significantly(P<0.01),with statistically significant differences;Compared with the middle dose group,the percentage of Th17 positive cells in PBMC of mice in low dose group and prednisone group increased significantly(P<0.01),and the percentage of Treg positive cells in PBMC of mice in low dose group decreased significantly(P<0.01),with statistically significant differences.6.The expression of RORγt and Foxp3 m RNA in PBMC of mice in each group showed that:Compared with the blank group,the expression level of RORγt m RNA in PBMC of mice in other groups increased significantly(P<0.01),and the expression level of Foxp3 m RNA decreased significantly(P<0.01),with statistically significant differences;Compared with the model group,the expression level of RORγt m RNA in PBMC of mice in each dose of traditional Chinese medicine group and prednisone group decreased significantly(P<0.01),and the expression level of Foxp3 m RNA increased significantly(P<0.01),with statistically significant differences;Compared with the high dose group,the expression level of RORγt m RNA in PBMC of mice in low dose group was significantly higher(P<0.01),and the expression level of Foxp3 m RNA in PBMC of mice in medium dose group,low dose group and prednisone group was significantly lower(P<0.01),with statistically significant differences;Compared with the middle dose group,the expression level of RORγt m RNA in PBMC of mice in the low dose group increased significantly(P<0.01),and the expression level of Foxp3 m RNA decreased significantly(P<0.01),with statistically significant differences.7.WB method was used to detect the expression of p-jak1/2/3,p-Stat3 and RORγt in PBMC of mice in each group.The results showed that: Compared with the blank group,the expression levels of p-jak1 in PBMC of other groups were up-regulated in varying degrees(P<0.05),while the expression levels of p-jak2/3,p-Stat3 and ror were significantly up-regulated(P<0.01),with statistically significant differences;Compared with the model group,the expression levels of p-jak1/2/3,p-Stat3 and RORγt in PBMC of mice in each dose group of traditional Chinese medicine and prednisone group were significantly decreased(P<0.01),with statistically significant differences;Compared with the high dose group,the expression levels of p-jak1/2/3 and p-Stat3 in PBMC of mice in medium dose group,low dose group and prednisone group increased in varying degrees(P<0.05),and the expression levels of RORγt in medium dose group and low dose group increased in varying degrees(P<0.05),with statistically significant differences;Compared with the middle dose group,the expression level of p-jak1/2/3 in PBMC of mice in the low dose group increased in varying degrees(P<0.05),with statistically significant differences.ICC method was used to detect the expression of p-jak1/2/3,p-Stat3 and RORγt in PBMC of mice in each group.The results showed that: Compared with the blank group,the expression levels of p-jak1/2,p-Stat3 and RORγt in PBMC of mice in other groups were up-regulated in varying degrees(P<0.05),and p-jak3 surface water was significantly up-regulated in medium dose group,low dose group and prednisone group(P<0.01);Compared with the model group,the expression levels of p-jak1/2/ 3,p-Stat3 and RORγt in PBMC of mice in each dose group of traditional Chinese medicine and prednisone group were significantly decreased(P<0.01);Compared with the high dose group,the expression levels of p-jak1/2/3 and RORγt in PBMC of mice in medium dose group,low dose group and prednisone group increased in varying degrees(P<0.05),and the expression levels of p-Stat3 in medium dose group and low dose group increased significantly(P<0.01);Compared with the medium dose group,the expression level of p-jak3 in PBMC in prednisone group decreased significantly(P<0.01),and p-Stat3 in low dose group increased significantly(P<0.05).Conclusion:1.Injection of anti platelet antibody can replicate the ideal ITP animal model.2.Modified Sijunzi Decoction can significantly improve thrombocytopenia in ITP mice,reduce the levels of IL-6,IL-17 and IL-22 in peripheral blood of ITP mice,and increase the level of TPO,GPⅡb/Ⅲa,IL-10 and TGF-β.3.the regulatory effect of Modified Sijunzi Decoction on the proportion of Th17/Treg cells in PBMC of ITP mice may be achieved by down regulating the expression of RORγt m RNA and up regulating the expression level of Foxp3 m RNA.4.Modified Sijunzi Decoction can inhibit the over activated Jak/Stat3 signal pathway in PBMC of ITP mice.5.Modified Sijunzi Decoction has obvious anti-inflammatory effect on ITP mice.Its mechanism may be realized by inhibiting the activation of Jak/Stat3 signal pathway,regulating the expression levels of RORγt,Foxp3 m RNA and protein,and then correcting the imbalance of Th17/Treg. |
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