| Objective:To explore the regulatory effect and mechanism of Xiao Wu Gui Decoction on immune cells of collagen-induced arthritis mice(CIA).Method:1 Xiao Wu Gui Decoction on the regulation of MDSCs,Th17 and Treg cells in CIA mice1.1 Construction and grouping of CIA mouse modelsCIA mice model was established and divided into model group(MO),methotrexate group(MT),Xiao Wu Gui Decoction Low-dose(XL),Medium-dose(XM),high-dose group(XH)and normal control group(Normal Control group,NC)was set,8 mice per group.Began to the 28th day,the XL,XM,and XH groups were given Xiao Wu Gui Decoction 19.5 g crude drug/kg/d,39 g crude drug/kg/d,75 g crude drug/kg/d;MT group was given methotrexate 2 mg/kg/w,the NC group and the MO group were given an equal dose of distilled water once a day,and administered orally.The medicinal solution was administered orally in an amount of 0.2 mL/10g for 28 consecutive days.1.2 The regulation effect of Xiao Wu Gui Decoction on MDSCs,Th17 and Treg immune cells in CIA miceDuring the experiment,the weight of the mice were weighed;the AI score was evaluated;the thickness of the mice foot was measured with a micrometer.After sampling,the Spleen Index(SI)was measured.The ratio of MDSCs in peripheral blood and MDSCs,Treg,Th17 cells in spleens were detected by flow cytometry.The ratio of MDSCs,Th17 and Treg cells was detected by flow cytometry.The correlation analysis between the ratio of MDSCs,Th17,Treg cells and AI score as well as the correlation analysis of MDSCs between the ratio of Th17,Treg cells were completed.Flow cytometry multi-factor detection technique was used to detect the expression levels of TNF-?,IL-6,IL-1?,IL-17 and IL-10 in plasma,HE staining was used to evaluate the pathological changes of the ankle joint,immunohistochemical staining was used to evaluate the expression levels of TNF-?,IL-1?,IL-17,Foxp3 in the ankle joint.2 Regulation effect of Xiao Wu Gui Decoction Lyophilized Powder on differentiation of mouse bone marrow cells into MDSCs,DC and macrophages2.1 CCK8 method was used to determine the effect of Xiao Wu Gui Decoction on the activity of mouse bone marrow cells and mouse bone marrow cells induced by GM-CSF.2.1.1 Effect of Xiao Wu Gui Decoction Lyophilized Powder on the activity of mouse bone marrow cells was observed.Mouse bone marrow cells were seeded on 96-well plates with 4000 cells per well and divided into 7 groups:NC group,0.25 mg/mL XWG group,0.5 mg/mL XWG group,1 mg/mL XWG group,1.25 mg/mL XWG group,2.5 mg/mL XWG group,5 mg/mL XWG group,and blank holes are set separately.Each group is provided with 5 duplicate wells.After 96 h,CCK8 method was used to detect cells activity.2.1.2 Effect of Xiao Wu Gui Decoction Lyophilized Powder on the activity of mouse bone marrow cells induced by GM-CSF was observed.Mouse bone marrow cells were seeded on 96-well plates with 4000 cells per well and divided into 8 groups:NC group,GM-CSF group,0.25 mg/mL XWG group,0.5 mg/mL XWG group,1 mg/mL XWG group,1.25 mg/mL XWG group,2.5 mg/mL XWG group,5 mg/mL XWG group,another blank well was set up,five duplicate wells were set for each group,and after 96 h,cell activity was detected by CCK8 method.2.2 Effect of Xiao Wu Gui Decoction Lyophilized Powder on mouse bone marrow cells differentiation into MDSCs,DC,macrophages2.2.1 Effect of Xiao Wu Gui Decoction Lyophilized Powder on mouse bone marrow cell differentiation into MDSCs,DC,macrophages was observed.Mouse bone marrow cells are seeded on 24-well plates with 10,000 cells per wells and divided into 5 groups:NC group,GM-CSF group,0.25 mg/mL XWG group,0.5 mg/mL XWG group,1 mg/mL XWG group.After 96h,the ratio of MDSCs,DC,macrophages was detected by flow cytometry.2.2.2 Effect of Xiao Wu Gui Decoction Lyophilized Powder on STAT3?MAPK and NF-?B signaling pathway on mouse bone marrow cells.Mouse bone marrow cells are seeded on cell culture dish with 10000000 cells per dish and divided into 3 groups:NC group,GM-CSF group,1 mg/mL XWG group.After 30 minutes,STAT3,ERK,JNK,p38MAPK and NF-?Bp65 phosphorylated protein levels by Western Blot method.Result:1 Study on the regulatory effect of Xiao Wu Gui Decoction on MDSCs,Th17 and Treg cells in CIA mice1.1 Compared with mice in MO group,AI scores and swelling degree of XM and XH groups were significantly reduced.1.2 Compared with mice in MO group,the ratio of G-MDSC in the peripheral blood of CIA mice in XM and XH group was significantly higher(p<0.05),and the ratio of M-MDSC was significantly decreased(p<0.01).The ratio of G-MDSC in the spleens of mice in XM and XH group was significantly higher(p<0.05),and the ratio of M-MDSC was significantly reduced(p<0.05).The proportion of Th17 cells in the spleens of mice in XM and XH group were significantly lower(p<0.01,p<0.01),and the proportion of Treg cells were significantly increased(p<0.01,p<0.01).1.3 The proportion of M-MDSC in peripheral blood of CIA mice was positively correlated with AI score(r=0.4008,p=0.0104).The proportion of M-MDSC in the spleens of CIA mice was positively correlated with AI score(r=0.4304,p=0.0056),the proportion of MDSCs and G-MDSC was negatively correlated with AI score(r=-0.4915,p=0.0013)(r=-0.3838,p=0.0145).The proportion of Th17 cells in the spleens of CIA mice was positively correlated with AI score(r=0.4092,p<0.0087),and the proportion of Treg cells was negatively correlated with AI score(r=-0.2732,p=0.0080).The proportion of M-MDSC in the peripheral blood was positively correlated with the proportion of Th17 cells(r=0.675,p<0.0001),the proportion of G-MDSC was positively correlated with the proportion of Treg cells(r=0.3604,p=0.0223).The ratio of M-MDSC in the spleens was positively correlated with the ratio of Th17 cells(r=0.4626,p=0.0026),and the ratio of MDSCs and G-MDSC was negatively correlated with the ratio of Th17 cells(r=-0.364,p<0.0209)(r=-0.6055,p<0.0001).1.4 Compared with mice in MO group,the expression levels of TNF-?,IL-6,IL-1?,and IL-17 in the plasma of mice treated with Xiao Wu Gui Decoction,and there was no significant difference in IL-10 expression levels.1.5 Compared with mice in MO group,ankle joint pathology scores,pannus scores,and bone destruction scores of mice in XM and XH groups were significantly lower(p<0.05),while ankle joint synovitis scores of mice in XH group was significantly lower(p<0.01).The expression levels of TNF-?,IL-1? and IL-17 in ankle joints of mice in XL,XM and XH groups were significantly reduced(p<0.05),and Foxp3 expression levels were significantly increased(p<0.05).2 Study on the regulation of Xiao Wu Gui Decoction Lyophilized Powder on the differentiation of mouse bone marrow cells into MDSCs,DC and macrophages2.1 When the concentration of Xiao Wu Gui Decoction Lyophilized Powder was 0.25 mg/mL and 0.5mg/mL,the viability of mouse bone marrow cells was higher than that of the NC group(p<0.01,p<0.01).When the concentration was 1 mg/mL,the cell viability was equivalent to that of the NC group.2.2 When the concentration of Xiao Wu Gui Decoction was 0.25 mg/mL and 0.5 mg/mL,the viability of mouse bone marrow cells induced by GM-CSF was significantly higher than that of the NC group(p<0.01,p<0.01).When the concentration was 1 mg/mL,the cell viability was equivalent to that of the NC group.2.3 Compared with GM-CSF group,after Xiao Wu Gui Decoction Lyophilized Powder interfered with GM-CSF-induced mouse bone marrow cells for 96 hours,the proportion of G-MDSC was significantly increased(p<0.01),but the proportion of M-MDSC and DC was significantly reduced(p<0.01).The proportion of macrophages was not significantly different in each group.2.4 Compared with GM-CSF group,after Xiao Wu Gui Decoction Lyophilized Powder interfered with GM-CSF-induced bone marrow cells for 30 minutes.The relative expression levels of STAT3,JNK,ERK,p38MAPK phosphorylated proteins of 1 mg/mL XWG group were significantly reduced(p<0.05,p<0.01,p<0.05,p<0.01).It was not significantly different in the relative expression of NF-?Bp65 phosphorylated protein in each group.Conclusion:1 Xiao Wu Gui Decoction can reduce the AI score,paw swelling and SI as well as inhibit the pathological damage of the ankle joint of CIA mice,thereby improving the severity of arthritis.2 MDSCs,Th17,Treg cells are related to the severity of arthritis in CIA mice.M-MDSC and Th17 cells may have pro-inflammatory effects,G-MDSC and Treg cells may have anti-inflammatory effects.MDSCs subsets may interact with Th 17 and Treg cells.M-MDSC may promote the differentiation of Th17 cells,and G-MDSC may promote the differentiation of Treg cells3 Xiao Wu Gui Decoction has the effect of regulating the balance of MDSCs subsets and Th17/Treg cells,increasing the levels of G-MDSC and Treg cells,reducing the levels of M-MDSC and Th17 cells,and promoting the interaction of MDSCs subsets with Th17 and Treg cells,thereby improving arthritis in CIA mice4 Xiao Wu Gui Decoction inhibits the expression of pro-inflammatory factors TNF-?,IL-6,IL-1?,IL-17 and promotes the expression of anti-inflammatory factor Foxp3,thereby improving arthritis in CIA mice.5 Xiao Wu Gui Decoction Lyophilized Powder can regulate the differentiation of mouse bone marrow cells,promote the differentiation of G-MDSC and inhibit the differentiation of M-MDSC and DC,which may be related to the inhibition of STAT3 and MAPK signaling pathway. |
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