Study On The Characteristics Of Inflammatory Factors In Joint Fluid Of Patients With Phlegm And Blood Stasis Syndrome And Liver And Kidney Deficiency Syndrome Of Knee Osteoarthriti

Objective:1.Protein antibody chip technology was used to measure the expression levels of inflammatory factors in joint fluid of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome,and to study the different characteristics of the expression level of inflammatory factors in joint fluid of patients with the above syndromes of knee osteoarthritis.2.Go enrichment analysis was used to explore the biological functions involved in the differential expression of inflammatory factors in joint fluid of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis;KEGG enrichment analysis was used to explore the possible signal pathway causing the differential expression of inflammatory factors in joint fluid of patients with Liverkidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis.3.Based on the above screened JAK/STAT signal pathway screened above,verify the detection results of protein antibody chip,and study the expression levels of JAK2 and STAT3 phosphorylated proteins(P-JAK2,P-STAT3)in joint cartilage,the concentration characteristics of MMP-13 and IL-23 in joint fluid and their correlation with the degree of syndrome in patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis.Methods:1.Clinical research(protein antibody chip detection):Using cross-sectional survey research methods,application of protein chip antibody technology,53 cases of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis were collected.The expression level of inflammatory factors was measured by protein chip antibody technology,and the imaging grade was quantified by K-L classification standard.The different characteristics of the expression level of inflammatory factors in joint fluid of the above syndrome were analyzed.2.Clinical research(enrichment analysis):Based on the differential expression of inflammatory factors obtained by protein antibody chip,GO enrichment analysis was applied to analyze the biological functions involved in the differential expression,and KEGG enrichment analysis was applied to analyze the signal pathways involved in the differential expression.3.Clinical research(validated based on JAK/STAT signal pathway results):The method of cross-sectional field investigation was used to collect articular fluid from 77 patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis.The expression levels of MMP-13 and IL-23 were measured by ELISA,the imaging degree was quantified by K-L grading standard,and the syndrome degree was quantified by TCM syndrome scoring scale,The expression levels of MMP-13 and IL-23 and their correlation with the degree of syndrome were analyzed.The gross morphology,pathological staining(H&E staining)and immunohistochemical staining(P-JAK2,P-STAT3)of articular cartilage in patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis were observed.Results:1.Expression characteristics of inflammatory factors in joint fluid of patients with Liverkidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis1.1 There was no significant statistical difference in the general situation and quantitative comparison of K-L grading standard between patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis(P>0.05);1.2 Protein antibody chip technology was used to obtain the expression level of inflammatory factors in joint fluid of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis.After analysis,differential inflammatory factors were obtained.The expression level of inflammatory factors in joint fluid of patients with Phlegm and Blood Stasis syndrome of knee osteoarthritis was higher than that of patients with Liver-kidney Deficiency syndrome(P<0.05);Specifically,it involved interleukin-related inflammatory factor(IL-1α,IL-1β,IL-2,IL-5,IL-10,IL-13),colony-stimulating factor-related inflammatory factors(G-CSF,GM-CSF,M-CSF),chemokines,interferon-associated inflammatory factors(CXCL-13,CCL-1),interferonassociated inflammatory factors(IFN-y),tumor necrosis factor associated inflammatory factor(TNF-α,TNF-β),growth factor associated inflammatory factor(PDGF-BB)(P<0.05);2.Study on the functional enrichment characteristics of the differential expression of inflammatory factors in joint fluid of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis2.1 There were 859 kinds of GO-BP terms significantly enriched by GO enrichment analysis.And 20 were selected by referring to Count and P values,as followed:response to molecule of bacterial origin,response to lipopolysaccharide,regulation of mononuclear cell proliferation,regulation of lymphocyte proliferation,regulation of leukocyte proliferation,regulation of B cell proliferation,positive regulation of response to external stimulus,positive regulation of peptidyl-tyrosine phosphorylation,positive regulation of mononuclear cell proliferation,positive regulation of leukocyte proliferation,positive regulation of leukocyte activation,positive regulation of immune effector process,positive regulation of hemopoiesis,positive regulation of cytokine production,myeloid leukocyte migration,mononuclear cell proliferation,leukocyte proliferation,leukocyte chemotaxis,cell chemotaxis,B cell proliferation.2.2 There were 32 kinds of GO-BP terms significantly enriched by GO enrichment analysis.And 20 were selected by referring to Count and P values,as followed:tumor necrosis factor receptor superfamily binding,tumor necrosis factor receptor binding,receptor ligand activity,Ras guanyl-nucleotide exchange factor activity,opioid receptor binding,interleukin-2 receptor binding,interleukin-1 receptor binding,interleukin-1 binding,guanyl-nucleotide exchange factor activity,growth factor receptor binding,growth factor binding,growth factor activity,G protein-coupled receptor binding,cytokine receptor binding,cytokine binding,cytokine activity,CXCR3 chemokine receptor binding,chemokine receptor binding,chemokine activity,CCR chemokine receptor binding.2.3 KEGG enrichment analysis showed that there are 35 differential signal pathways with significant enrichment.And 20 were selected by referring to Count and P values,as followed:JAK/STAT signaling pathway,PI3K-Akt signaling pathway,NF-kappa B signaling pathway,MAPK signaling pathway,Viral protein interaction with cytokine and cytokine receptor,Type I diabetes mellitus,Thl and Th2 cell differentiation,T cell receptor signaling pathway,Rheumatoid arthritis,Intestinal immune network for IgA production,Inflammatory bowel disease,IL-17 signaling pathway,Hematopoietic cell lineage,Graft-versus-host disease,Cytokine-cytokine receptor interaction,Chagas disease,Autoimmune thyroid disease,Asthma,Allograft rejection,African trypanosomiasis.3.To explore the syndrome characteristics of Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis based on JAK/STAT signal pathway,and the correlation between the syndrome degree and the syndrome characteristics.3.1 There was no significant difference in general situation and quantitative comparison of K-L grading standards between patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis(P>0.05).3.2 The concentrations of MMP-13 and IL-23 in joint fluid of patients with Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis were higher than those of patients with Liver-kidney Deficiency syndrome(P<0.05).3.3 There was a positive correlation between the concentration of MMP-13 in joint fluid and TCM syndrome score in patients with Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis(r2=0.121,P<0.05).There was a positive correlation between the concentration of IL-23 in joint fluid and TCM syndrome score in patients with Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis(r2=0.278,P<0.05).3.4 There was a positive correlation between the concentration of MMP-13 in joint fluid and TCM syndrome score in patients with Liver-kidney Deficiency syndrome of knee osteoarthritis(r2=0.001,P=0.826).The concentration of IL-23 in joint fluid of patients with Liver-kidney Deficiency syndrome of knee osteoarthritis was positively correlated with TCM syndrome score(r2=0.102,P<0.05).3.5 There was obvious differences in the gross morphology and HE staining characteristics of knee cartilage tissue in patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis sy ndrome of knee osteoarthritis;3.6 The levels of JAK2 and STAT3 phosphorylation in immunohistochemical tissues of knee cartilage in patients with Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis was higher than that in patients with Liver-kidney Deficiency syndrome of knee osteoarthritis.Conclusions:1.Clinical research results showed that(characteristics of differential inflammatory factors):1.1 The increased expression of concentration in pro-inflammatory(nociceptive)inflammatory factors in joint fluid of patients with knee osteoarthritis(IL-1α、IL-1β、IL-5、IL-7、GM-CSF、M-CSF、G-CSF、CCL-1、TNF-α、TNF-β、IFN-γ)was the syndrome characteristic of Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis.1.2 Decreased concentration of anti-inflammatory(protective)inflammatory factors(IL2,IL-10,IL-13,CXCL13 and PDGF-BB)in the joint fluid of patients with knee osteoarthritis is the syndrome characteristic of Liver-kidney Deficiency syndrome of knee osteoarthritis.1.3 The different characteristics of the expression levels of inflammatory factors in joint fluid of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis supported the scientificity of "different syndromes of the same disease"and "different treatments of the same disease".2.Clinical study results(feature of functional enrichment):2.1 Through GO enrichment analysis,multiple differential molecular functions and biological processes can be obtained.The inflammatory factors annotated contain both proinflammatory and anti-inflammatory effects,which were in line with the characteristics of TCM syndromes in which evil and positive qi compete.2.2 Among the biological signal pathways obtained by KEGG enrichment,the enhanced expression of pro-inflammatory response related signal pathways was the syndrome characteristic of Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis,and the decreased expression of anti-inflammatory response related signal pathways is the syndrome characteristic of Liver-kidney Deficiency syndrome of knee osteoarthritis.2.3 The enrichment results of protein antibody chip in articular fluid of patients with Liverkidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis showed that the syndrome characteristics are different,and there were different biological process regulation in "harmful biological process" and "protective biological process",suggesting that different syndromes have different biological basis under the same disease,so as to provide biological basis for "different treatment of the same disease" in traditional Chinese medicine.3.Clinical study results(verified based on JAK/STAT signal pathway results):3.1 The concentration characteristics of MMP-13 and IL-23 in joint fluid of patients with Liver-kidney Deficiency syndrome and Intermingled Phlegm and Blood Stasis syndrome of knee osteoarthritis were different.The concentrations of MMP-13 and IL-23 in joint fluid of patients with Intermingled Phlegm and Blood Stasis syndrome can reflect the severity of their syndrome,and the concentration of IL-23 in joint fluid of patients with Liver-kidney Deficiency syndrome can reflect the severity of their syndrome.3.2 The general morphological observation,H&E staining,JAK2 and STAT3 phosphorylation immunohistochemical staining,combined with the concentration characteristics of MMP-13 and IL-23,it was speculated that the difference of syndrome characteristics between Intermingled Phlegm and Blood Stasis syndrome and Liver-kidney Deficiency syndrome of knee osteoarthritis may be related to the different activation degree of JAK2-STAT3 signal pathway.The above characteristics were consistent with the etiology and pathogenesis analysis of TCM under the syndrome of this disease,which provided an objective basis for the physical basis of the syndrome and enriched the scientific connotation of syndrome identification.