The Mechanism Of Action Of Astragalus, Angelica And Honeysuckle In Relieving Inflammation In Mice With Collagen-induced Arthriti

Objective:To investigate the mechanism of the combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae in promoting the inflammation resolution and repairing joint destruction of collagen-induced arthritis(CIA)by regulating the function of type 2 innate lymphocytes(ILC2s).Methods:Bovine type Ⅱ collagen(CⅡ)was emulsified with complete Freund’s adjuvant(CFA)and incomplete Freund’s adjuvant(IFA),respectively.The emulsifiers were used for the first immunity and enhanced immunity of DBA/1 mice,respectively,to construct CIA mice model simulating the pathological characteristics of rheumatoid arthritis(RA).The combination of Radix Astragali and Radix Angelicae Sinensis,Caulis Lonicerae alone,the combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae were used for 28 days of drug intervention,and leflunomide tablets were used as positive control drugs.The metatarsophalangeal joint thickness and clinical score of mice were recorded once a week.The components of the three herbs were analyzed by high performance liquid chromatography electrospray ionization/mass spectrometry(HPLC-ESI/MSn).The pathological changes of CIA mice joints were observed by hematoxylin-eosin(HE),safranin O fast green and tartrate-resistant acid phosphatase(TRAP)staining.Proliferation and differentiation of Naive T cells,Effector T cells,Memory T cells,Thl 7 cells,Treg cells and ILC2s cells were detected by flow cytometry.The distribution and polarization of M1-type and M2-type macrophages were observed by immunofluorescence labeling of macrophages with F4/80,INOS and CD206 antibodies.The secretion of pro-inflammatory cytokines IL-17,IFN-y and anti-inflammatory cytokines IL-4 were detected by enzyme-linked immunosorbent assay(ELISA).The expressions and phosphorylation of key molecules of the Janus tyrosine kinase/signal transducer and activator of transcription(JAK/STAT)signaling pathway,including JAK2,JAK3 and STAT6 were measured by Western-blot.Results:(1)Pharmaceutical ingredient:Seven constituents of Radix Astragali,six constituents of Radix Angelicae Sinensis and eleven constituents of Caulis Lonicerae were identified by HPLC-ESI/MSn.(2)Joint morphology:The results showed that after 28 days of drug treatment,the thickness of metatarsophalangeal joints of CIA mice in all drug groups was significantly decreased(P<0.01),and the clinical score was significantly decreased(P<0.01).(3)Pathological staining:HE staining showed that there were a lot of inflammatory cell infiltration,synovial hyperplasia,cartilage destruction and bone erosion in the joints of CIA mice in model group.Safranin O fast gree staining showed that the structure of articular cartilage and subchondral bone in CIA mice was damaged,with slight or no safranin O staining,and the loss score of safranin O staining was increased(P<0.01).Trap staining showed that the expression of purplish red osteoclasts on the bone surface was significantly increased,and the number of osteoclasts in bone tissue was increased(P<0.01).After 28 days of drug treatment,synovial hyperplasia and cartilage destruction were alleviated,bone destruction was slowed down,the loss score of safranin O staining was decreased(P<0.01),and the number of osteoclasts was decreased(P<0.01).(4)Flow cytometry:Results showed that after 28 days of drug treatment,the percentage of Naive T cells(CD4+CD44LowCD62L+)in each drug group was significantly decreased(P<0.01).The percentage of effector T cells(CD4+CD44LowCD62L-)was significantly increased after intervention of leflunomide and the combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae(P<0.01),and was significantly increased after intervention of Caulis Lonicerae and the combination of Radix Astragali and Radix Angelicae Sinensis(P<0.05).The percentage of memory T cells(CD4+CD44HighCD62L-)was significantly increased after leflunomide intervention(P<0.01),and was significantly increased after intervention of the combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae(P<0.05),but there was no significant difference in the combination of Radix Astragali and Radix Angelicae Sinensis group(P>0.05)and Caulis Lonicerae group(P>0.05).Compared with normal group,the percentage of Th17 cells(CD4+IL17+)in model group was significantly increased(P<0.01),the percentage of Treg cells(CD4+CD25+Foxp3+)was significantly decreased(P<0.01),and the percentage of ILC2s cells(LIN-KlRG1+CD127+CD278+)was significantly decreased(P<0.01).After 28 days of drug intervention,compared with the model group,the percentage of Th17 cells in all drug groups was significantly decreased(P<0.01),the percentage of Treg cells was significantly increased(P<0.01),and the percentage of ILC2s cells was significantly increased(P<0.01).(5)Immunofluorescence:The results showed that INOS antibody expression was obvious in CIA mice in model group,and CD206 antibody expression was obvious in normal group and drug groups.Drug intervention can promote the polarization of macrophages from M1 type(F4/80+INOS+CD206-)to M2 type(F4/80+INOS-CD206+),and the promoting effect of the combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae group is better than the combination of Radix Astragali and Radix Angelicae Sinensis group and Caulis Lonicerae group.(6)ELISA:The results showed that after 28 days of drug treatment,compared with the model group,the secretion of pro-inflammatory cytokines IL-17 and IFN-y in CIA mice in drug groups was significantly decreased(P<0.01),and the secretion of anti-inflammatory cytokines IL-4 was significantly increased(P<0.01).(7)Western-blot:The results showed that compared with the normal group,JAK2 and JAK3 protein expression and phosphorylation level of CIA mice in model group were significantly increased(P<0.01).After 28 days of drug treatment,these abnormally increased protein expression and phosphorylation level of CIA mice in all drug groups were significantly decreased(P<0.01).The decreased STAT6 expression and phosphorylation level were significantly increased after drug intervention(P<0.01).Conclusions:The combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae can alleviate synovial inflammation,repair articular cartilage destruction and reduce bone damage in CIA mice.The main mechanism may be related to promoting the proliferation and differentiation of ILC2s,promoting the polarization of macrophages from M1 type to M2 type,and regulating the balance of immune network.Therefore,promoting the proliferation and differentiation of ILC2s can promote inflammation resolution,and ILC2s can serve as a potential therapeutic target for RA.In addition,the synergistic effect of the three drugs was better than the effect of the combination Radix Astragali and Radix Angelicae Sinensis,and the effect of Caulis Lonicerae alone.The combination of Radix Astragali,Radix Angelicae Sinensis and Caulis Lonicerae can provide a choice for the prescription of RA.