Mechanism Research Of Xinfeng Capsule On Pulmonary Function Based On The Cross-Talk Of TGF-β1/Smads And ERK Pathway In A Rat Model Of Adjuvant Atrhritis

1ObjectiveBased on the Traditional Chinese Medicine theory of "correlationbetween lung and spleen ",“Lung Paralysis" as the breakthroughpoint,this paper analyzes and summarizes medical pathogenesis of lungfunction damage in rheumatoid arthritis (RA). From the perspective ofanimal experiment to observe the dynamic changes of paw swelling,arthritis index (AI) and lung function changes in adjuvant arthritis (AA)rat, detects changes of TGF-β1/Smads, ERK signaling pathways andcytokine in AA rats’ synovial and lung tissue, evaluates influence ofChinese medicine Xinfeng Capsule (XFC), and explores the mechanismof XFC improves lung function in AA rats.2Methods2.1Theoretical studyCollecting and organizing the relevant literature of RA lung disease,screening and summarize the research status of the RA lung diseasepathogenesis,evaluation and analysis of transforming growth factor beta1(TGF-beta1) induced by Smads, ERK pathway activation pathways.Studied from a theoretical point of the relationship between lung functionreduce in RA and the TGF-β1/Smads, ERK signaling pathway cross-talk. This article based on the basic theories of TCM, discussed therelationship between TCM "lung" and "spleen", by studying from theperspective of physiological pathology and etiology pathogenesis, andexplained the mechanism of “jianpihuashi Tongluo therapy” in thetreatment of RA decline in lung function.2.2Experimental Study50Wistar rats rats were randomly divided into five groups:normalcontrol (NC) group, model control (MC) group, methotrexate (MTX)group, Tripterygium glycosides piece (TPT) and XFC group, n=10. Inaddition to the NC group, right rear foot plantar skin of the rest ratsinjected Freund’s complete adjuvant (CFA)0.1ml of proinflammatorymodeling, strengthen the immune by injection in the base of the tail ofCFA0.05ml at the15d, dosage at the19d. Dosage groups are asfollows: the XFC group:0.034g/1ml/100g, TPT group:0.57mg/1ml/100g,MTX group0.095mg/1ml/100g, NC、MC group:physiological saline9mg/1ml/100g; XFC,TPT,NC,MC group once a day, MTX group once aweek, each group continuous medication for30d. Observed the rats’body mass, paw swelling, arthritis index (AI) and pulmonary functionchanges, morphological changes of lung tissue, synovium by lightmicroscopy and electron microscopy, tested the expression of cytokineTGF-β1in interleukin the prime (IL)-1β, IL-4, IL-10, gamma interferon(IFN-γ), connective tissue growth factor (CTGF), fibroblast growth factor(FGF) by enzyme-linked immunosorbent assay, detected changes ofSmads/ERK pathway in synovial membranes and lung tissue by PCR,immunohistochemistry and Western blot.3Results3.1The theoretical research results3.1.1TGF-β1/Smads pathway activated prompted RA lung function decreased.TGF-β1can first combined TβR Ⅱ (its receptor) to form atrimer complex TβRI, than further phosphorylated Smad2/3, p-Smad2/3to separate from TβRI, which than combined to form complexes withSmad4, carried signals from the cytoplasm into the nucleus to activatetranscription, promote lung lesions; whereas Smads7is inhibitorymolecules, if reduceing Smad7inhibition ability, inhibition oftranscription of Smads signals cannot be lost, the inhibitory effect on RAlung lesions, resulting in decreased lung function.3.1.2ERK signaling pathway induced by RA lung function decreased:the activation of the ERK pathway may mediates cell growth,differentiation, proliferation, promotes the secretion of fibroblasts andexcessive accumulation of extracellular matrix (ECM) in pulmonaryinterstitial and alveolar, makes the alveolar distance increased to forminterstitial lung, decreases in pulmonary capillary bed and blood flow,changes in blood rheology, reduces pulmonary diffusion, weakens theexchange of oxygen and carbon dioxide, while the activation of ERKpathway can regulate the expression of inflammation-related genes,leading to lung function decreased.3.1.3Smads and ERK pathway cross-talk is an important factor in thedecline in RA lung function: the activation process of TGF-β1/Smadsignaling pathway is also affected by the regulation of the ERK pathway,Smad2/3contains ERK phosphorylation sites,which can be phosphoryl-ated by ERK, promote p-Smad2/3and Smad4in binding andtranscription. Meanwhile, ERK pathway is regulated by Smads pathway,Smad4regulates transcription of ERK pathway related cytokines orkinase, which activates ERK signaling pathway, Smads and ERKpathway cross-talk common led to the decline in lung function.3.1.4The "Spleen asthenia" is the pathological basis of RA decline in lung function: spleen asthenia caused by Qi and blood deficiency,spleen can not bear the lung,lead to deficiency of lung-qi and yin,andalso lead to failure of pulmonary qi to disperse and descend，resulting insuch syndrome like: cough, shortness of breath, spontaneousperspiration,etc; asthenia of splenic qi,failure of transportation andtransformation, which fails to distribute fluid and leads to attack of fluidretention on the lung, then lung fails to its Regulatory functions,manifeasting in such syndrome like: cough with profuse whitish sputum,even asthmatic breath with sputum rale, chest pain, prolonged andincurable; lung collaterals block, see more lung texture disorder,nodules, interstitial fibrosis were frequent in spleen deficiency caused byphlegm and blood stasis.3.1.5The “Jianpihuashitang Tongluo Therapy” makes significantimprovement in RA lung function: According to the TCM pathogenesis ofRA decline in lung function “asthenia of spleen and stomach, asthenia ofqi and blood, accumulation of fluid and dampness, phlegm and bloodstasis " and “inclusion of asthenia and sthenia”characteristics, clinicallydefered to "eliminating pathogen and strengthening vital qi " principle oftreatment, using the " Jianpihuashitang Tongluo Therapy ", applyedXFC treatment of RA and pulmonary dysfunction, and achieved goodresults, XFC could further improve pulmonary symptoms and signs, toimprove the dispersion function and ventilation function by improvingjoint and systemic symptoms and signs.3.2The experimental study results3.2.1Changes of lung function in rats and the impact of XFCCompared with NC group, the MC rats lung function FEV1, FEF50, FEF75,MMF, PEF decreased (P <0.01).Compared with the MC group, FEV1, FEF50, FEF75, MMF, PEF were increased in the XFC group (P <0.05or P <0.01).Compared with the MTX group, the level of FEF50, FEF75MMF wereincreased in the XFC group (P <0.05); compared with TPT group,pulmonary function parameters FEF75, MMF were increased in the XFCgroup (P <0.05or P <0.01).3.2.2Changes of paw swelling, AI in rats and the impact of XFCCorrelation analysis showed the AA rat lung function FEF75, pawswelling were negative correlation, FEF50, FEF75and the AI werenegatively correlated (P <0.05or P <0.01).Prior to administration, compared with the NC group, body weight wasreduced in MC rats, and paw swelling degree, AI were significantlyhigher than those the MC rats (P <0.01).30days after administration, compared with the NC group, toe swelling,AI were elevated in MC rats; compared with the MC group, rat pawedema degrees and AI were lower in each treatment group; comparedwith the MTX group (P <0.05or P <0.01).3.2.3Changes of joint morphological in rats and the impact of XFCLight microscopic observation: Compared with NC group, the MC ratssynovium and its affiliated organizations visible to a large number ofinflammatory cell infiltration increased vascular increased synoviallayered, showed villous projections embedded in the intra-articularsurface of the articular cartilage exfoliative or missing such as. Electronmicroscopy: the MC group synovial cells deformation, swelling ofmitochondria, rough endoplasmic reticulum was reduced, damaged,incomplete nuclear membrane, the uneven distribution of chromatin,irregular or partial disappearance of cristae arranged.The XFC treatment, the rat synovial visible small number of neutrophilsand other inflammatory cell infiltration, embedded in the intra-articular part of the synovial tissue, the articular surface was orderly; synovialcells the mitochondrial small amount of deformation, swelling, nuclearmembrane boundary more clearly, chromatin more uniform, most of thecristae arranged complete. Compared with the MTX group the XFCgroup synovial cells reduce mitochondrial swelling; the XFC group TPTgroup no significant difference.3.2.4Changes of lung tissue morphologic in rats and the impact of XFCLight microscopy showed that: compared with the NC group, MC grouprat alveolar structure was irregular, the alveolar cavity atrophy ordisappear, was part of the lung substantive changes in lung intervalswidened visible in the interstitial infiltration of inflammatory cells, alveolarepithelial cell proliferation; reduce the number of electron microscopy Ⅱalveolar epithelial cells, the membrane structure was not completemitochondrial swelling, degeneration, reduced lamellar bodies wereemptying state, interstitial lung fibroblasts.XFC treated rats compared with regular alveolar structure, part of thealveolar space atrophy, deformation, seen in a small number ofneutrophils and other inflammatory cells in the interstitial infiltration;basic integrity of the structure of type Ⅱ alveolar epithelial cells,abundant rough endoplasmic reticulum, mitochondria and large partiallyintact, occasionally lamellar bodies emptying phenomenon; comparedwith the MTX group, the the XFC group of lung tissue inflammatory cellinfiltration and decrease in alveolar epithelial cells; the TPT groupshowed no significant differences.3.2.5Changes of serum cytokine in rats and the impact of XFCCorrelation analysis showed AA the rats lung function FEV1, FEF75IL-1β, IL-4was negatively correlated FEF25, FEF50, FEF75, MMF withTGF-β1, CTGF, FGF negatively correlated; PEF and IL-10were positively correlated, FEV1, FEF50were positively correlated withTh1/Th2,FEF50, FEF75and IFN-γ were positively correlated (P <0.05orP <0.01).Compared with the NC group, the level of IL-1β, IL-4, TGF-β1, CTGF ofserum were increased in MC, IFN-γ, IL-10, Th1/Th2cells, FGF werereduced (P <0.05or P <0.01).Compared with the MC group, the expression of IL-1β, IL-4, TGF-β1,CTGF were reduce, and the expression of IFN-γ, IL-10, Th1/Th2, FGFwere increased in the XFC group (P <0.05or P <0.01).Compared with the MTX group, CTGF was decreased, and IFN-γ,expression of IL-10, Th1/Th2were increased in XFC group (P <0.05);Compared with TPT group, TGF-β1were decreased in the XFC group(P <0.05).3.2.6Changes of Smads pathway in rats and the impact of XFCCorrelation analysis showed FEV1and TGF-β1mRNA, protein werenegatively correlated, FEF25and Smad4protein were negativelycorrelated, FEF50and TβRⅡ protein expression were negativelycorrelated with FEF75and TGF-β1, Smad3mRNA, p-Smad2/3proteinwas negatively correlated with PEF and TGF-β1protein were negativelycorrelated (P <0.05); FEF50, MMF Smad7mRNA were positivelycorrelated(P <0.05).Compared with NC group, the level of TGF-β1, Smad2/3/4mRNA,TGF-β1, TβRI, TβRⅡ, Smad2/3, p-Smad2/3, Smad4protein wereincreased, expression of Smad7mRNA, protein of synovial lung tissuewere decreased in MC group (P <0.05or P <0.01).Compared with the MC group, expression of Smad2, Smad3, Smad4mRNA and TGF-β1, TβRI, TβRⅡ, Smad2/3, p-Smad2/3protein weredecreased, expression of Smad7mRNA and protein of lung tissue were elevated in XFC group (P <0.05or P <0.01).Compared with the MTX group, expression of TGF-β1,Smad3mRNAand TGF-β1, Smad2/3protein were decreased, Smad4mRNA TβRIprotein were reduced, Smad7mRNA was increased in XFC group (P<0.05); Compared with TPT group, Smad3mRNA, TGF-β1, p-Smad2/3, Smad4protein were reduced, Smad7protein was increased in theXFC group (P <0.05), p-Smad2/3of lung tissue was lowered, Smad7mRNA was increased in the XFC group (P <0.05).3.2.7Changes of ERK1/2in rats and the impact of XFCCorrelation analysis showed pulmonary function FEV1and ERK2mRNAwere negatively correlated, FEF25and p-ERK1/2protein werenegatively correlated, FEF75, MMF and ERK1/2, p-ERK1/2proteinwere negatively correlated (P <0.05). ERK1mRNA, Smad3mRNA inTβRⅡ protein of lung tissue were positively correlated, ERK2mRNA andp-Smad2/3was positively correlated, ERK1/2proteins, Smad2mRNA,Smad4protein were positively correlated, p-ERK1/2proteins andSmad4mRNA, p--Smad2/3protein were positively related (P <0.05);p-ERK1/2protein and Smad7were negatively correlated (P <0.05).Compared with the MC group, expression of ERK2mRNA, p-ERK1/2protein of lung tissue were decreased, ERK1, ERK2mRNA, ERK1/2,p-ERK1/2protein were decreased in the XFC group (P <0.05or P<0.01).Compared with the MTX group, expression of ERK1, ERK2mRNA oflung tissue were decreased in the XFC group (P <0.05).4Conclusions4.1Changes of lung function in AA rats4.1.1Pulmonary dysfunction features: changes of pulmonary functionparameter FEV1, MMF, PEF, FEF50, FEF75were reduced in AA rats, which mainly characterized ventilatory dysfunction, and accompanied bya certain degree of small airway function damage.4.1.2Smads and ERK pathway cross-talk lead to reduce lung function:Smads and ERK pathway can inducte TGF-β1, which graduallyactivated by phosphorylation, and then enter the nucleus involved intranscriptional activity, resulting in AA lung lesions occur, pulmonaryThe function was reduced.4.1.3TCM pathogenesis characteristics:"Spleen" in RA lung functionoccurred, plays an important role in the development, temper deficiency,lack of righteousness, lung weakness; temper deficiency sputum Thewet endogenous governance section of lung failure; temper deficiency,phlegm, pulmonary collaterals block.4.2Efficacy of XFC on AA rats4.2.1XFC can inhibit joint inflammation reaction of AA rats: XFC toreduce the the AA rat paw swelling and AI, to reduce the infiltration ofinflammatory cells of the synovial alleviate local congestion and swelling,inhibit the formation of pannus, improve joint function.4.2.2XFC can improve the level of AA rats lung function by improvinglung function parameters FEV1, FEF50, FEF75, MMF, PEF expression:XFC improve the AA rat pulmonary ventilation function and small airwayfunction, enhanced pulmonary gas exchange rate and improve lungfunction level.4.2.3XFC can improve the quality of the AA rats: XFC Jianpiyiqi thedampness Tongluo the efficacy, can significantly improve the the AArats appetite and water intake, increase the level of body mass, improvesystemic symptoms and functional activity.4.2.4XFC can adjust the AA rat lung tissue morphology significantly:XFC can reduce the degree of infiltration of inflammatory cells, inflammatory mediators by inhibiting lung tissue stimulation, reduce theAA rat pulmonary interstitial fibrosis, maintain alveolar type Ⅱ cellfunction and morphology of the organelle.4.3Mechanisms of XFC improve lung function4.3.1XFC can reduce the AA rats joint inflammation reaction to improvelung function: XFC seeping through the inhibition of inflammatory cellinfiltration of synovial tissue, reduce the systemic inflammatoryresponse, reducing inflammatory mediators and the stimulation of lungtissues and organs, inhibition of interstitial lung disease occur, improvelung function level.4.3.2XFC can reduce the AA rats lung inflammation to improve lungfunction: XFC by the anti-inflammatory effect, reducing the directstimulation of inflammatory mediators of alveolar promote absorption ofneutrophils and other inflammatory cells or clear, to reduce lunginflammation reaction, improve lung function level.4.3.3XFC can adjust the AA rats cytokine balance to improve lungfunction: XFC FGF expression by up-regulating IL-10, IFN-γ, loweredIL-1β, IL-4, TGF-β1, CTGF expression, inhibition of AA rats Theincidence of pulmonary fibrosis to improve lung function level.4.3.4XFC can improve the AA rat lung tissue morphology to improvelung function by reducing the inflammatory exudate lung tissue damage,keep the lung tissue pathomorphological maintain alveolar type Ⅱepithelial cell structure, increased alveolar ventilation function: XFCimprovement in lung function and diffusion capacity.4.3.5XFC can adjust AA rats TGF-β1/Smads pathway to improve lungfunction: XFC increase Smad7expression, inhibition of p-Smad2/3with Smad4binding and prevents Smad2/3phosphorylation, delayingthe the AA rat lung fibrosis the occurrence, and improve lung function. 4.3.6XFC can inhibit the AA rats ERK1/2pathway expression toimprove lung function by inhibition of ERK1/2phosphorylation process,preventing the activation of the signaling pathway, reducing fibrogenicfactors on lung tissue damage.4.3.7XFC can adjust Smads and ERK pathway cross-talk to improvelung function: XFC reduce signaling and DNA transcription activity,inhibiting the formation of the AA rat pulmonary interstitial fibrosis byregulating signaling pathways cross-talk to improve lung function.