| 1 Objective Based on the guidance of Xin'an Medicine(treat illness from the spleen,tonify the spleen and resolve dampness to dredge the channel),laboratory rats with adjuvant arthritis(AA)are expected to be used for studying the immunity reaction of rheumatoid arthritis,oxidative stress status,changes of pulmonary function and regulatory mechanism that Xinfeng capsule(XFC)brings to the rheumatoid arthritis lung disease.Those subjects are also used to explore immunological mechanism of that XFC treats rheumatoid arthritis.Discuss the function of the cross talk of Notch/Treg and ROS-PKC/NF-?B signal pathway makes the pulmonary function reduced when infecting rheumatoid arthritis.That provides evidences for treating rheumatoid arthritis by traditional Chinese therapy.2 Research background RA related lung function decline not only correlated with Notch signaling pathway mediated differentiation and development of immune reactive cells,such as regulatory T cells(Treg)which are directly involved in the process of tissue fibrosis,but also correlated with oxidative stress injury induced by Reactive Oxygen Species(ROS).Notch/Treg signaling pathway activation can reduce lung function of patients with rheumatoid arthritis,while ROS-PKC/NF-?B signaling pathway induced oxidative stress injury can affect lung function as well.And the existence of crosstalk between the two signaling pathways may be one of the mechanisms leading to the reduction of RA lung function.Through long-term clinical practice summary,Xin'an doctors summed up the dialectical thought that treating from the spleen in the treatment of arthralgia.The treatment method is widely used in clinical treatment of rheumatism and rheumatoid-related lung disease.That excessive dampness due to deficiency in spleen,deficiency of qi and blood,phlegm and phlegm-blood stasis are important symptom of syndrome,and excessive dampness due to deficiency in spleen,phlegm and blood stasis obstructing lung is an important pathogenesis of rheumatism-related lung disease characteristics.Therefore,fortifying the spleen and replenish qi,transforming into dampness,free the collateral vessels and making earth engenders metal is an important treatment method for curing RA related lung disease.3 Methods Our research intended to use the adjuvant arthritis(adjuvant arthritis,AA)rats for the model.Rats' lung function,the joints,lung tissues morphological changes were detected.Using flow cytometry instrument to detect AA rat peripheral blood expression frequency of Treg cells.Using Elisa method to detect the rat serum levels of cytokines and the expression of oxidative stress indicators.Using immunohistochemical method to detect AA rats' CD4 and FOXP3 expressions in lung tissue.Realtime PCR and Western blot method were used to detect Notch/Treg and ROS-PKC/NF-?B signaling pathway related gene and protein expression changes in the lung tissue.Than we evaluate the effect of XFC treatment on AA rats;discuss the role of cross talk between Notch/Treg and ROS-PKC/NF-?B signaling pathways on the pathogenesis of RA related lung disease.4 Results 4.1 The efficacy of XFC on AA rats: XFC can increase the body quality of AA rats,reduce the AA rats' foot metatarsus swelling degree and arthritis index(P < 0.05 or P < 0.01).Comparison between groups of AA rats after treatment,and compared with LEF control group(P < 0.05 or P < 0.01),XFC mid-dose group of high quality,toes swelling degree no statistical difference;Compared with AGA in the control group,the toes swelling degree and AI in XFC mid-dose group significantly decreased(P < 0.01 or P < 0.05).XFC also can improve AA rats' joint pathological structure.4.2 Effects of XFC on AA rats' pulmonary function: comparison between groups of AA rats' lung function parameters.Compared with NC group,MC group of FVC,FEV1 pulmonary function parameters were significantly decreased(P < 0.05).Compared with MC group,the treatment groups rats' lung function parameters have a certain degree of increase(P < 0.01 or P < 0.05).Pulmonary function of mid-dose XFC group improved significantly higher on AA rats,much better than LEF group rats and AGA control group rats(P < 0.01 or P < 0.05).XFC also can improve AA rats' lung pathological structure.4.3 Effects of XFC on AA rats' blood serum cytokines: Compared with NC group,IL-6,IL-17 significantly raised and IL-10,IL-35 significantly reduced(P < 0.05 or P < 0.01)in MC group.Compared with MC group,IL-6,IL-17 expression were reduced,while IL-10,IL-35 increased in all the treatment groups(P < 0.05 or P < 0.01).Among the high,middle and low doses XFC groups,the mid-dose XFC group showed higher IL-10,IL-35 and lower IL-6,IL-17(P < 0.05 or P < 0.01).Compared with LEF control group,XFC group showed higher IL-17,IL-35 and lower IL-10(P < 0.05 or P < 0.01),but IL-6 showed no statistical difference.Compared with AGA control group,XFC group showed higher IL-35 and lower IL-6,IL-17(P < 0.05 or P < 0.01);but IL-10 has no statistical difference.4.4 Effects of XFC on AA rats' serum oxidative stress indexes: compared with NC group,the levels of NO and i NOS in peripheral blood of MC group rats were significantly increased,and the contents of NADPH and HIF-1 were decreased(P<0.05 or P <0.01).Compared with the MC group,the expression of NO and i NOS was significantly decreased in the treatment group,and the levels of NADPH and HIF-1 were significantly increased(P<0.05 or P<0.01).Among the high,middle and low doses XFC groups,the levels of NO,i NOS were lower and the levels of NADPH and HIF-1 were higher in middle dose XFC group(P <0.05 or P <0.01).Compared with the LEF control group,the levels of NADPH and HIF-1 in the XFC group were significantly higher than those in the control group.The levels of NO in the XFC group were significantly lower than those in the control group(P <0.05 or P <0.01),but there was no significant difference in i NOS.Compared with AGA control group,the content of i NOS in XFC middle dose group was significantly lower(P <0.01),and there was no significant difference in NO,NADPH and HIF-1. 4.5 Effects of XFC on peripheral blood Treg cells in AA rats: Compared with NC group,CD4+CD25+Notch1+Treg and CD4+CD25+Notch3+Treg expression were decreased in MC group(P <0.01).Compared with MC group,CD4+CD25+Notch1+Treg and CD4+CD25+Notch3+Treg expression were increased in each treatment group(P <0.05 or P <0.01).Compared with LEF control group,the expression of CD4+CD25+Notch3+Treg was slightly lower in XFC group(P <0.05),and the number of CD4+CD25+Notch1+Treg was not statistically different.Compared with AGA control group,the number of CD4+CD25+Notch1+Treg in XFC middle dose group was significantly higher(P<0.01),and there was no significant difference in CD4+CD25+Notch3+Treg.4.6 Effects of XFC on Notch/Treg signaling pathway in lung tissue of AA Rats: Immunohistochemistry showed that CD4 expression were increased and Fox P3 expression were decreased in lung tissue of MC group(P <0.05 or P <0.01).Compared with MC group,CD4 expression were decreased and Fox P3 expression were increased in XFC group(P <0.05 or P <0.01).Compared with the LEF control group and the AGA control group,the expression of CD4 in the middle dose XFC group was lower and the Fox P3 was significantly higher(P <0.05 or P <0.01).Realtime-PCR was used to detect the m RNA expression of Receptor Notch1,ligand delta1 and target gene transcription factor HES1 in AA rats' lung tissue.Compared with NC group,the expression of Notch1,delta1 and HES1 m RNA in Notch pathway of MC group was significantly increased.After drug treatment,the expression of three proteins was significantly reduced.Among the three doses XFC groups,the expression of Notch1,delta1 and HES1 m RNA was significantly higher in middle dose XFC group than that other XFC group(P <0.05 or P <0.01).Compared with the LEF control group,the expression of Notch1 in the XFC middle dose group was significantly lower than that in the control group(P <0.05),and the expression of HES1 was not significantly different.Compared with AGA control group,the expression of Notch1,delta1 and HES1 m RNA was significantly lower in XFC middle dose group(P <0.05 or P <0.01).Western Blot was used to detect the expression of Notch pathway-associated protein in AA rats' lung tissue.Compared with NC group,the expression of Jagged 1,Jagged 2 and Notch 4 in Notch pathway was significantly increased in MC group,and the expression of three proteins was significantly reduced after drug treatment.Among the three doses XFC groups,the expression of Jagged 1,Jagged 2 and Notch 4 protein was significantly decreased in the XFC middle dose group(P<0.05 or P<0.01).Compared with the LEF control group,the reduction of Notch4 in the XFC middle dose group was weaker than that of the LEF group(P<0.05),and there was no significant difference in Jagged 1 and Jagged 2 expression.Compared with the AGA control group,the expression of Jagged 1,Jagged 2 and Notch 4 was significantly lower in the XFC middle dose group(P <0.05 or P <0.01).4.7 Effects of XFC on ROS-PKC/NF-?B signaling pathway in lung tissue of AA Rats Immunohistochemistry showed that the expression of PKC and NF-k B in lung tissue of MC group were significantly increased(P<0.05 or P<0.01).Compared with MC group,PKC and NF-k B expression were decreased in each treatment group(P<0.05 or P<0.01).Compared with the LEF control group,the level of NF-k B in the middle dose XFC group was weaker(P<0.05),and the degree of PKC was better than that of LEF(P<0.05).Compared with AGA control group,the levels of PKC and NF-k B were significantly decreased in middle dose XFC group(P <0.05 or P <0.01).Realtime-PCR was used to detect the m RNA expression of PKC,NF-?B and target gene Rac-1.Compared with NC group,the expression of ROC pathway PKC,NF-?B and target gene Rac-1 m RNA in MC group was significantly increased.After treatment,the expression of three proteins was significantly reduced.Among the high,middle and low dose of XFC groups,the middle dose XFC group can effectively reduce the expression of PKC,NF-?B and Rac-1 m RNA(P<0.05 or P<0.01).Compared with the LEF control group,the level of Rac-1 m RNA in XFC middle dose group was weaker(P<0.05),and there was no significant difference in PKC and NF-?B.Compared with AGA control group,the decrease of PKC,NF-?B and Rac-1 m RNA in XFC middle dose group was significantly decreased(P <0.01)Western Blot was used to detect the expression of ROS pathway-associated protein in AA rats' lung tissue.The expression of Rac-1,PKC-? and NF-k B p65 in MC group was significantly increased.After treatment,the expression of three proteins was significantly reduced.Among the high,middle and low dose of XFC groups,the expression of Rac-1,PKC-? and NF-k B p65 protein was significantly decreased in the middle dose XFC group(P <0.05 or P <0.01).Compared with the LEF control group,the levels of Rac-1 and NF-k B p65 in the XFC middle dose group were weaker(P<0.05),but there was no significant difference in the reduction of PKC.Compared with AGA control group,the decrease of Rac-1,PKC-? and NF-k B p65 protein in XFC middle dose group was significantly decreased(P <0.01).4.8 the relationship between AA rats' lung tissue Notch/Treg signaling pathway,ROS-PKC/NF-?B signaling pathway and lung function parameters Spearman correlation analysis shows that: AA rat pulmonary function parameters FEV1 was negatively correlated with Notch1 m RNA(P < 0.05),FEF75 was negatively correlated with HES1 m RNA(P < 0.05).FEF25 was negatively correlated with Jagged2,Notch4 protein(P < 0.05),FEF75,MMF negatively correlated with Jagged1,Notch 4 protein respectively(P < 0.05).AA rat pulmonary function parameters FEV1,FEF75 were negatively correlated with NF-?B and target genes Rac1 m RNA(P < 0.05),FEF50 was negatively correlated with PKC m RNA(P < 0.05).FEF25,FEF75 were negatively correlated with NF-?B protein(P < 0.05),FEF75 was negatively correlated with PKC protein(P < 0.05),FEF25 was negatively correlated with Rac-1 protein(P < 0.05).The Notch1 m RNA of Notch pathway in lung tissue of AA rats showed a positive correlation with NF-?B,Rac-1 m RNA and Rac-1protein(P < 0.05).HES1 m RNA showed a positive correlation with PKC protein(P < 0.05).Notch4 and PKC protein m RNA were positively correlated(P < 0.05).Jagged1 protein were positively correlated with PKC protein(P < 0.05).Jagged2 m RNA were positively correlated with Rac-1 m RNA and PKC protein(P < 0.05).5 Conclusion Xinfeng capsule can not only improve the body weight of AA rats,improve the swelling of the foot of rats and reduce the arthritis index,but also can improve the synovial membrane of the joint structure,improve synovial inflammation and inhibit puerperal hyperplasia.It can also improve the lung function of AA rats and lung tissue pathological structure.The mechanism may be related as follows: 5.1 XFC can relieve arthritis and improve pulmonary function parameters XFC can bring relief to arthritis sufferers of AA rats,and improve the pulmonary function parameters,thus to improve joint symptoms and lung function.5.2 XFC can improve lung tissue pathological structure Xinfeng capsule can regulate the immune response,improve oxidative stress,reduce the inflammation of the joints and lung tissue,and improve the pathological structure of lung tissue,thus to improve lung function.5.3 XFC can enhance the antioxidant capacity and improve the state of peroxidation,thus to reduce oxidative stress injury in lung tissue Xinfeng capsule can increase the level of antioxidant substances such as NADPH,HIF-l,and reduce NO and i NOS levels which reduce to inhibit redox reaction.XFC can effectively regulate the oxidative stress state and reduce lung tissue oxidative stress injury.5.4 XFC can rebalance the cytokine network,relieve synovial inflammation,thus to reduce immune inflammatory injury in lung tissue Xinfeng capsule can increase the anti-inflammatory cytokines such as IL-10,IL-35,reduce the proinflammatory cytokines such as IL-17,IL-6.It can inhibit the proinflammatory effect of cytokines,enhance anti-inflammatory ability and inhibit inflammatory factor-mediated vascular endothelial dysfunction.Thereby it can effectively improve lung tissue immune inflammatory injury and have anti-inflammatory anti-immune effect.5.5 XFC can regulate the crosstalk between Notch/Treg signaling pathway and ROS-PKC/NF-?B signaling pathway,thus to regulate immune effector cells Xinfeng capsule can increase the level of Treg cells in peripheral blood of AA rats.It can also inhibit the over-activation of Notch/Treg signaling pathway and ROS-PKC/NF-?B signaling pathway,inhibit the immune inflammatory response in tissue,and improve the antioxidant capacity,reduce oxidative stress injury,thus to improve lung function. |
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