The Mechanism Of Jianpi Qingre Tongluo Prescription Regulating LncRNA HOTAIR/APN/PI3K/AKT Combination To Improve Self-perception Patient With Osteoarthritis Damp-heat Obstruction Syndrome

1 Objective Explore jianpi qingre tongluo prescription(JQP)through the non-coding RNA HOTAIR/adiponectin/pho sphatidylinositol 3-kinase/protein kinase B(lncRNA HOTAIR/APN/PI3K/AKT)combination on self-perception of patient(SPP),immune inflammation and lipid metabolism in osteoarthritis(OA)patients with damp-heat obstruction syndrome.2 Methods2.1 Study 1: based on retrospective data mining,the change of self-perception of patient with damp-heat obstruction syndrome in OA and intervention of iianpi qingre tongluo prescription Self-controlled test was used to collect jianpi qingretongluo prescription(JQP),including xinfeng capsule(XFC,patent No.: ZL201310011369.8),huangqin qingre chubi capsule(HQC,Patent No.: ZL201110095718.X)peripheral blood mononuclear cells(uclear cells,PBMCs)of OA patients and healthy people before and after treatment,SPP scale and laboratory indexes of OA patients with damp-heat obstruction were prepared: complement(C)3,C4,immunoglobulin(Ig)A,Ig G,Ig M,hypersensitive C-reactive protein(hs-CRP),erythrocyte sedimentation rate(ESR),total Cholesterol(TC),triglyceride(TG),high density liptein cholesterol(HDL-C),low-Density Lipoprotein Cholesterol(LDL-C);real time quantitative PCR(RT-q PCR)was used to detect the expression of lncRNA HOTAIR.Enzyme-linked immuno sorbent assay,ELISA was used to detect interleukin(IL)-10,tumor necrosis factor(TNF)-α,adiponectin(APN),and necrosis factor.Correlation analysis,association rule analysis and Logistics regression were used to analyze the correlation between SPP and lncRNA HOTAIR,immune inflammation and lipid metabolism indexes.Using the receiver-operating characteristic curve(ROC)analysis lncRNA HOTAIR effect to the diagnosis of OA.Association rules were used to analyze the correlation between JQP and SPP,lncRNA HOTAIR,improvement of immune inflammation and lipid metabolism.2.2 Study 2: based on interference or overexpression of LncRNA HOTAIR,studing the mechanism of jianpi qingre tongluo prescription medicated serum regulating LncRNA HOTAIR/APN/PI3K/AKT combination to improve the inflammatory response of OA chondrocytes The medicated serum of jianpiqingretongluo prescription(JQP)was prepared,cell models of OA-PBMCs and OA chondrocytes(CH)were co-cultured in Transwell cells,and lncRNA HOTAIR interference or overexpression plasmid was constructed and transfected into co-cultured cells.cell counting kit-8(CCK-8)was used to detect cell viability,and PBMC stimulation concentration and optimal concentration drug-containing serum were screened.The expression of lncRNA HOTAIR was detected by RT-q PCR.Using ELISA method to detect the expressions of IL-4,IL-6,IL-10 and IL-1β.Western blotting(WB)was used to detect phosphorylated(p)-PI3 K,p-AKT,PI3 K,AKT,PTEN,APN,ADIPOR1 and ADIPOR2 proteins.After transfecting lncRNA HOTAIR overexpression plasmid,JQP drug-containing serum was added for intervention to observe the changes of cell viability,PI3K/AKT signaling pathway,cytokine levels,APN and its receptors.3 Results3.1 Study 1: based on retrospective data mining,the change of self-perception of patient with damp-heat obstruction syndrome in OA and intervention of jianpi qingre tongluo prescription.(1)Changes of SPP,lncRNA HOTAIR expression,inflammation and lipid metabolism indexes in OA patients with damp-heat obstruction syndrome: SPP indexes in OA patients with damp-heat obstruction syndrome: damp-heat obstruction syndrome score,SAS,SDS and VAS scores increased abnormally,while BP,SF,PF,RP,GH,VT and MH scores decreased abnormally.Indexes of immune inflammation and lipid metabolism: ESR,hs-CRP,Ig A,Ig G,Ig M,C3,C4,LDL-C,TC,TG were abnormally increased,while HDL-C was abnormally decreased.Compared with normal control group,lncRNA HOTAIR expression was increased in PBMCs of OA patients with damp-heat obstruction syndrome(P<0.05),the cytokine IL-10 was decreased,the TNF-α was increased(P<0.05),and the lipid metabolism index: APN was decreased(P<0.05).(2)ROC curve analysis of lncRNA HOTAIR in damp-heat obstruction syndrome of OA: lncRNA HOTAIR can be used as a molecular marker to assist in the diagnosis of OA(AUC=83.20%,P<0.0001,95%CI: 0.7371-0.9249).(3)Correlation analysis of OA damp-heat obstruction syndrome SPP and lncRNA HOTAIR,immune inflammation and lipid metabolism indexes: There is a significant correlation between SPP and immune inflammation indexes: VAS and ESR,Lg G were positively correlated(P<0.05),PF and TG were negatively correlated(P<0.05),RP and Ig A,Ig G was negatively correlated(P<0.05),GH and Ig G was negatively correlated(P<0.05),VT was negatively correlated with the ESR(P<0.05).RE was negatively correlated with C4 and TNF-α(P<0.05),damp-heat obstruction syndrome score was positively correlated with TC,TG and HDL-C(P<0.05),SAS was positively correlated with hs-CRP and TNF-α(P<0.01),SDS was positively correlated with ESR(P<0.01).lncRNA HOTAIR was significantly correlated with SPP and immunoinflammatory indexes :Ig A,hs-CRP and TNF-α were positively correlated with lncRNA HOTAIR(P<0.05 or P<0.01).(4)Association rule analysis of SPP in damp-heat obstruction syndrome of OA,immune inflammation and lipid metabolism indexes: The SPP index SAS,PF,VAS,RE,the score of damp-heat obstruction syndrome,RP and ESR,TNF-α,IL-10,hs-CRP had a support degree greater than 35%,confidence degree greater than 60%,and improvement degree greater than 1 in patients with OA damp-heat obstruction syndrome,indicating that SPP was strongly correlated with immunoinflammatory indexes and cytokines.According to the results of network diagram,SPP,immune inflammation and lipid metabolism indicators have strong correlation in OA patients with damp-heat obstruction syndrome.(5)Logistics regression analysis of OA damp-heat obstruction syndrome SPP and lncRNA HOTAIR,immune inflammation and lipid metabolism indexes: Under the conditions of B>0,P<0.05,OR>1,SAS was strongly correlated with HDL-C,TNF-α,ESR,VT and lncRNA HOTAIR,SDS and C4(P<0.05).Therefore,lncRNA HOTAIR,HDL-C,TNF-α,ESR and C4 are risk factors for SPP.(6)Effects of JQP on SPP,lncRNA HOTAIR,immune inflammation and lipid metabolism indexes of OA with damp-heat obstruction syndrome: Compared with patients with damp-heat obstruction syndrome of OA before treatment,SPP indexes of JQP after treatment are as follows: SAS,SDS,VAS scores decreased(P<0.05),BP,SF,PF,RP,VT,MH scores increased(P<0.05).lncRNA HOTAIR expression decreased(P<0.05);Immunoinflammatory indexes: ESR,hs-CRP,Ig A,Ig G,Ig M,C3,C4 scores decreased(P<0.05);Cytokines: IL-10 was increased and TNF-α was decreased(P<0.05);Lipid metabolism index: decrease LDL-C,TC and TG;increase HDL-C and APN(P<0.05).(7)Association rule analysis of JQP and improvement of SPP,lncRNA HOTAIR,immune inflammation and lipid metabolism indexes in damp-heat obstruction syndrome of OA: the confidence of JQP and SPP,immune inflammation and lipid metabolism indexes were all greater than 50%,the support degree was greater than50%,and the improvement degree was greater than 1,indicating that JQP was strongly correlated with SPP,immune inflammation and lipid metabolism(P<0.05).3.2 Study 2: based on interference or overexpression of LncRNA HOTAIR,studing the mechanism of jianpi qingre tongluo prescription medicated serum regulating LncRNA HOTAIR/APN/PI3K/AKT combination to improve the inflammatory response of OA chondrocytes(1)The optimal proportion and time of OA-CH co-culture model stimulated by OA-PBMCs,and the optimal concentration and time of action of JQP drug-containing serum: The optimal dosage ratio of OA-CH stimulated by OA-PBMCs was 3:1,and the optimal time was 48h;20% serum concentration containing JQP was the best,and the best action time was 48 h.(2)Impact of lncRNA HOTAIR interference or overexpression on cytokine expression.In the pc DNA3.1-lncRNA HOTAIR group,IL-1β and IL-6 expressions were significantly higher than in the pc DNA3.1-NC group,however IL-4 and IL-10 expressions were significantly lower(P<0.05).In the si-lncRNA HOTAIR group,IL-1βand IL-6 expressions were dramatically downregulated compared to the si-NC group,whereas IL-4 and IL-10 expressions were significantly upregulated(P<0.05).(3)The impact of interference or overexpression of lncRNA HOTAIR on PI3K/AKT signaling pathway.In order to determine the effect of lncRNA HOTAIR on PI3K/AKT pathway,western blots analysis was done to looking for the expression of PI3 K,p-PI3 K,AKT,p-AKT,APN,ADIPOR1,ADIPOR2,and PTEN,overexpression of the lncRNA HOTAIR resulted in decreased expression of APN,ADIPOR1,and PTEN and increased expression of p-PI3 K and p-AKT(P<0.05).On the contrary,lncRNA HOTAIR’s interference has led to the opposite trend.Nevertheless,PI3 K,AKT,and ADIPOR2 expression were unaffected significantly by the overexpression or silencing of the lncRNA HOTAIR.(4)LncRNA HOTAIR regulates the effect of APN in co-culture cells on PI3K/AKT signaling pathway,cell viability and cytokine levels.The results showed that overexpression of APN had no discernible influence on the level of lncRNA HOTAIR,but promoted the activity of chondrocytes,suppressed PI3 K and AKT phosphorylation,lowered pro-inflammatory factors,and increased anti-inflammatory factors.Overexpression of lncRNA HOTAIR can reverse this effect.In conclusion,the role of APN in promoting cell viability,inhibiting phosphorylation of PI3K/AKT signaling pathway,reducing pro-inflammatory factors and promoting anti-inflammatory factors expression may be regulated by lncRNA HOTAIR.(5)The effect of jianpi qingre tongluo prescription drug-containing serum on lncRNA HOTAIR expression: RT-q PCR results showed that the drug-containing serum of JQP could reduce the expression of lncRNA HOTAIR(P <0.05).(6)The effect of jianpi qingre tongluo prescription drug-containing serum on cytokines in co-culture cells by lncRNA HOTAIR: ELISA results showed that JQP drug-containing serum could reduce the expression of IL-6 and IL-1β(P<0.001),and increase the levels of IL-4 and IL-10(P<0.001).(7)Effects of jianpi qingretongluo prescription medicated serum on APN expression level and PI3K/AKT signaling pathway through lncRNA HOTAIR: After transfection of pc DNA3.1-lncRNA HOTAIR,the expression of PTEN,APN,ADIPOR1 and ADIPOR2 was increased,while the protein levels of p-PI3 K and p-AKT were decreased(P<0.05).These findings suggested that the JQP might suppress the PI3K/AKT signaling pathway and lower the level of lipid metabolism and inflammatory indexes through lncRNA HOTAIR.4 Conclusion(1)OA with damp-heat obstruction syndrome experience worse,which is manifested as increased damp-heat obstruction syndrome score,VAS,SAS,SDS;Decreased BP,SF,PF,RP,GH,VT,MH scores,up-regulated lncRNA HOTAIR expression,increased immunoinflammatory indexes,abnormal lipid metabolism indexes.SPP was significantly correlated with lncRNA HOTAIR,immune inflammation and lipid metabolism.(2)JQP can not only improve the expression of lncRNA HOTAIR,immune inflammation and lipid metabolism indexes in OA damp-heat obstruction syndrome,but also significantly improve SPP.(3)The medicated serum of JQP can inhibit the inflammatory response of co-culture cells and enhance cell vitality by regulating the combination of lncRNA HOTAIR/APN/PI3K/AKT,thus achieving the purpose of improving the disease condition.