Data Mining Research On Oxidative Stress In Actue Gouty Arthritis Improved By Jian Pi Li Shi Qing Re Tong Luo Therapy And The Effect On AMPK Signal Pathway

1 Objective From the three aspects of data mining,clinical experiment and in vitro cell experiment to observe the oxidative stress,inflammatory reaction,quality of life,AMPK signaling pathways and related laboratory index changes and the effects of HQC on them of patients with AGA,and based on AMPK signal path to explore the mechanism of HQC improve oxidative stress and inflammation in patients with AGA.2 Methods2.1 Research on clinical data mining Using the Case acquisition system software of the first affiliated hospital of Anhui university of traditional Chinese medicine to collect the patients who were hospitalized on our hospital from May 2012 to October 2017 who were the patients with AGA(wet and heat accumulation type).The clinical data mining system developed by the information center of our hospital is applied.The general information of the patients in the hospital was collected,and information such as the medical treatment and laboratory examination results were collected too,and established the "Anhui provincial hospital rheumatology data center".The above data were analyzed using SPSS 22.0 and SPSS Clementine 11.1 software Aprior module.2.2 Clinical Research Observe the age,gender,course of disease,joint and systemic symptom score,life quality score and other laboratory indicators of the 60 AGA.ELISA method was used to detect the expression level of peripheral blood serum's IL-6,IL-4,IL-10,TNF-?,IL-1?,SOD,MDA,LPO,T-AOC,PPAR?,CD36,AMPK,FoxO3 a,p-FoxO3 a,MnSOD.The expression of CD36 was detected by flow cytometry.The expression of PPAR?,AMPK,FoxO3 a was detected by RT-PCR.The expression of PPAR?,AMPK,FoxO3 a,P-FoxO3 a and MnSOD was detected by Western Blotting.TheWestergren method was used to detect ESR.Using Hitachi HITACHI7060 automatic biochemical analyzer to detect other biochemical indexes;Blood routine was detected by automatic blood analyzer(XT2000).The integral value of symptoms and signs of AGA patients(joint,body,etc.),VAS integral and other integral values were observed by questionnaire.SPSS22.0 statistical software was used to analyze the relationship between oxidative stress,inflammatory response indicators and above indexes.The 60 hospitalized patients were divided into the experimental group by the random number table(30 cases of HQC group,30 cases)and the control group(30 cases of meloxicam group).Observed two groups indicators sucs as oxidative stress related indicators(SOD,TAOC,MDA,LPO),joint and systemic symptoms and signs integral value,VAS score,cytokines(IL-1?,TNF-?,IL-6,IL-4,IL-10),AMPK signaling pathway,discuss the improvement of indicators before and after the treatment.2.3 Experimental study in vitro To collect the anticoagulant blood of AGA patients,and the PBMC was isolated.Adding totally 1640 medium containing serum,put in 37? and 5%CO2 incubator,After the cells were adhered to the wall for 24 h.adding different doses of HQC,Collecting cells after 24 hours,extraction of RNA,Fluorescence quantitative PCR was used to detect the expression of PPAR?mRNA,AMPK-?1mRNA,FoxO3 amRNA and MnSODmRNA,in peripheral blood monocytes of AGA patients.The expression of PPAR?,AMPK?1,p-AMPK?1,FoxO3 a,p-FoxO3 a and MnSOD protein was detected in the peripheral blood monocytes of AGA patients by the western blotting method(WB).At the same time,the expression levels of IL-1?,IL-4,LPO,MDA,SOD and TAOC were detected by ELISA.3 Results3.1 Results of clinical data mining of 598 patients with AGA.3.1.1 Usage of traditional Chinese medicine There were more than 300 Chinese herbs in 598 patients.The top 20 Chinese traditional Chinese medicine(TCM),which has the highest frequency,is concentrated in the fivemajor categories of the use of spleen dampness medicinal,heat-clearing medicinal,wind-dampnessdispelling medicinal,lood-activating and stasis-resolving medicinal,qi-tonifying medicinal,qi-regulating medicinal.3.1.2 Correlation analysis of medicine.The correlation between the two medicines' efficacy was explored by applying association rules,and the association was made between spleen dampness medicinal,heat-clearing medicinal,wind-dampness dispelling medicinal,lood-activating and stasis-resolving medicinal,qi-tonifying medicinal,qi-regulating medicinal.Minimum support and confidence are 20% and 70% respectively.The three medicine with the highest confidence are:wind-dampness dispelling medicinal and heat-clearing medicinal(100%),spleen dampness medicinal and wind-dampness dispelling medicinal(100%),spleen dampness medicinal and heat-clearing medicinal(99.83%).The highest frequency of 20 flavor drugs were associated,The three medicine with the highest confidence are:Tao Ren paired with Hong Hua(98.47%),the Shan Yao paired with Yi Yi Ren(94.94%),and the Ze Lan paired with Ze Xie(94.17%).3.1.3 Cluster analysis of medicine.SPSS 22.0 was used for cluster analysis of medicines with high frequency.It was found that four medicine,such as spleen dampness medicinal,heat-clearing medicinal,lood-activating and stasis-resolving medicinal and the wind-dampnessdispelling medicinal,were used to treat the AGA.3.1.4 Analysis of association rules between laboratory index and traditional Chinese medicine.According to the analysis of Aprior module,it was concluded that SOD increased with Yi Yi Ren,hs-CRP,?1-AGP and ESR decreased with Yi Yi Ren,UA decreased with Che Qian Cao,with high degree of confidence and support.3.2 Clinical results3.2.1 Changes of oxidative stress,cytokines and signaling pathways in AGA patients.Compared with the healthy control group,the Various parameters of patients in AGA were obviously abnormal.The indicator of the body's ability to resist oxidation,Such as SOD and TAOC,was significantly lower than that of healthy control group.The indicator of the body's peroxidation,Such as MDA and LPO,were significantly higher than those in the healthy control group(P<0.01);the cytokines of patients in AGA were significantly abnormal.Among them,the pro-inflammatory(IL-1?,TNF-?,IL-6)were significantly increased in the healthy control group,and the anti-inflammatory factor(IL-4,IL-10)were significantly lower in the healthy control group(P<0.01).the indicator of the AGA patients was significantly abnormal.The expression of PPAR?,CD36,AMPK,FoXO3 a,p-FoXO3 a and MnSOD was significantly lower than that in healthy control group(P<0.05 or P<0.01).3.2.2 Changes in expression of PPAR?,AMPK and FoxO3 a genes in patients with AGA.There was a decrease in the expression of PPAR?mRNA,AMPK-?1mRNA and FoxO3 a mRNA in AGA patients(P<0.01).3.2.3 Changes in expression of PPAR?,AMPK,FoxO3 a,p-Foxo3 a and MnSOD in the peripheral blood of AGA patients.The expression of PPAR?,AMPK-?1,FoxO3 a,p-FoxO3 a and MnSOD in AGA patients were lower than those in the healthy control group(P<0.05 or P<0.01).3.2.4 Changes of expression levels of CD36 in peripheral blood monocytes of AGA patients.The fluorescence intensity of CD36 was significantly lower than that in the healthy control group(P<0.05 or P<0.01).3.2.5 Changes in other laboratory indicators of AGA patients.Compared with the healthy control group,the AGA patients had various immune indicators,inflammatory markers and other abnormalities(P<0.05 or P<0.01).3.2.6 Correlation analysis of oxidative stress index and cytokines and pathway indexes in AGA patients.Correlation analysis showed that SOD was negatively correlated with TNF-?,positively correlated with AMPK,PPAR? and MnSOD,and TAOC was positively correlated with AMPK and CD36,and MDA was negatively correlated with IL-10,AMPK,FoXO3 a,p-FoxO3 a,PPAR?,MnSOD,and positively correlated with IL-1?.LPO was negatively correlated with FoXO3 a,p-FoxO3 a,MnSOD and CD36(P<0.05,P<0.01).3.2.7 Correlation analysis of cytokines and pathway indexes in AGA patients.Correlation analysis showed that AMPK was positively correlated with IL-6 and negatively correlated with TNF-?.FoXO3 a was negatively correlated with TNF-?.p-FoXO3 a was negatively correlated with TNF-?.PPAR?was positively correlated with IL-4 and IL-10,and was negatively correlated with TNF-?.MnSOD was positively correlated with IL-4 and IL-10,and negatively correlated with TNF-?.CD36 was positively correlated with IL-4 and IL-10(P<0.05,P<0.01).3.2.8 Correlation analysis of oxidative stress index and some laboratory indexes in AGA patients.Correlation analysis showed that SOD was negatively correlated with UA,ESR and hs-CRP,and was positively correlated with IgA.TAOC is negatively correlated with ESR and hs-CRP;It was positively correlated with CD36,and MDA was positively correlated with ?1-AGP.LPO was positively correlated with hs-CRP(P<0.05,P<0.01).3.2.9 The correlation analysis between the oxidative stress index and symptom quantization score and VAS score in AGA patients.Correlation analysis showed that LPO was positively correlated with joint pain(P<0.05,P<0.01).3.2.10 Effects of HQC on the clinical efficacy of AGA patients.After treatment,the total effective rate of the control group and the treatment group was90% and 96.7%,and the total effective rate of the treatment group was higher than that of the control group(P<0.05).The efficiency of the treatment group(60.0%)was significantly higher than that in the control group(43.3%)(P<0.05).3.2.11 Changes of oxidative stress indexes,cytokines and path-related indexes were compared between the two groups.Compared with before treatment,both groups of SOD and TAOC were both increased,and MDA and LPO both decreased(P<0.01 or P<0.05).Compared with the two groups,the increase of TAOC in the treatment group was more obvious and the decrease of LPO was more obvious(P<0.05).both groups of IL-4 and IL-10 increased,and IL-6,IL-1?,and TNF-? were all decreased(P<0.01 or P<0.05).After treatment,the increase of IL-4and IL-10 in the treatment group was more obvious,and the decrease of IL-6,IL-1?,and TNF-? was more significant(P<0.01 or P<0.05).both groups of PPAR?,CD36,AMPK,FoXO3 a,p-Foxo3 a and MnSOD all increased(P<0.01).Compared with the two groups,the increase of PPAR?,CD36,FoXO3 a,p-Foxo3 a and MnSOD was more obvious in the treatment group(P<0.01).3.2.12 Effects of HQC on the expression of PPAR?mRNA,AMPK-?1mRNA and FoxO3 amRNA in peripheral blood of AGA patients.Compared with pre-treatment,the gene expression levels of both PPAR?mRNA,AMPK-?1mRNA and FoxO3 amRNA were all increased(P<0.01 or P<0.05).Compared with the two groups,the level of gene expression in the treatment group of PPAR?mRNA,AMPK-?1mRNA and FoxO3 amRNA was significantly increased(P<0.01).3.2.13 Effects of HQC on the expression of PPAR?,AMPK-?1,FoxO3 a,p-FoxO3 a and MnSOD in peripheral blood of AGA patients.The protein expression levels of PPAR?,AMPK-?1,FoxO3 a,p-FoxO3 a and MnSOD were all increased in the two groups compared with before treatment(P<0.01 or P<0.05).After two groups of treatment,the expression level of PPAR?,AMPK-?1,FoxO3 a,p-FoxO3 a and MnSOD increased significantly in the treatment group(P<0.01).3.2.14 Effects of HQC on the expression level and fluorescence intensity of monocyte CD36 in peripheral blood of AGA patients.Compared with pre-treatment,the relative fluorescence intensity of both groups wasincreased(P<0.01).After treatment,the relative fluorescence intensity of CD36 was significantly higher in the treatment group(P<0.01).3.2.15 Effects of HQC on other laboratory indexes of AGA patients.Compared with pre-treatment,both groups could improve the immunological,inflammatory and oxidative stress indexes of AGA patients(P<0.01 or P<0.05).After two groups of treatment,the treatment group was better than the control group(P<0.01 or P<0.05)in improving the patients' partial immunity,inflammation and oxidative stress.3.2.16 Effects of HQC on TCM symptom score and VAS score in AGA patients.Compared with the pre-treatment phase,both groups could improve the TCM symptom score and VAS score in the AGA patients(P<0.01 or P<0.05).Compared the two groups after treatment,the treatment group in improving patients with Chinese medicine symptom integral(joint pain,joint fever,adverse joint flexion and extension)and VAS score is better than that in control group(P<0.01 or P< 0.05).3.3 Experimental results in vitro.3.3.1 Effects of HQC medicated serum on the cytokines and oxidative stress indexes of PBMC in AGA patients.Compared with the normal group,IL-1?,LPO and MDA in the other groups were higher than the normal group,and IL-4,SOD and TAOC were all higher than the normal group(P<0.01).Compared with HQC group,LPO of blockers,MDA of HQC+blockers were higher(P<0.05),SOD and TAOC of HQC+blockers group were lower(P<0.01),compared with the model group,the IL-1?,LPO and MDA in some groups were lower.IL-4,SOD,TAOC were higher(P<0.05 or P<0.01).3.3.2 Effects of HQC medicated serum on PPAR?mRNA,MnSODmRNA,AMPK-?1mRNA and FoxO3 amRNA of PBMC in AGA patients.Compared with normal group,the rest of the group PPAR?mRNA,MnSODmRNA,AMPK-?1mRNA,FoxO3 amRNA expression were lower than normal group(P<0.01),compared with HQC group,the above-mentioned indexes of HQC+blocker group andblockers group were lower(P<0.05 or P<0.01),compared with the model group,the above indexes of HQC group,HQC+blocker group were higher,and the index of blocker group were lower than those of model group(P<0.05 or P<0.01).3.3.3 Effects of HQC medicated serum on the expression of MnSOD,AMPK-?1,p-AMPK?1,PPAR?,FoxO3 a and p-FoxO3 a in the PBMC of AGA patients.Compared with the normal group,MnSOD,AMPK-?1,p-AMPK?1,PPAR?,FoxO3 a,p-FoxO3 a protein expression were all lower than normal group(P<0.01).Compared with HQC group,the above-mentioned indexes of HQC+blocker group and blocker group were lower(P<0.01),compared with the model group,the above indexes of HQC group,HQC+blocker group were higher,and the index of blocker group were lower than those of model group(P<0.01).4 ConclusionsThere is an oxidative stress state in the AGA patients,which is characterized by a decrease in the antioxidant capacity of the body and is closely related to the inflammatory response.HQC can improve the clinical symptoms of AGA patients,at the same time,it can also significantly improve the oxidative stress and inflammatory response status of AGA patients.In addition,the clinical efficacy and improvement of oxidative stress and inflammatory response status were better than that of meroxicam control group.The mechanism of HQC improving oxidative stress and inflammatory response in AGA patients is as follows:(1)HQC by raising the anti-inflammatory cytokine(such as IL-4,IL-10),cut proinflammatory cytokines(such as TNF-?,IL-6,IL-1?),control inflammation,restore the balance of the organism oxidation and anti-oxidation and improve clinical symptoms.(2)HQC increased the expression of AMPK signaling pathway by stimulating PPAR? and improved the antioxidant capacity of patients,thus improving the effect of oxidative stress and inflammatory response in patients.