Study On The Mechanism Of Qingre Huayu Gel In The Treatment Of Rheumatoid Arthritis Based On The Key Molecules Of Glucose Metabolism Reprogramming

BackgroundRheumatoid arthritis(RA)is a common immune disease with high disability rate.Joints are the main target organs invaded by RA.The pathological mechanism of active RA synovial microenvironment is complex.Immune metabolism,especially glucose metabolism reprogramming is the focus of research.Targeted glucose metabolism reprogramming is a new strategy of RA therapy.Traditional Chinese medicine has significant advantages in the treatment of RA.Qingre Huayu gel,formerly known as "compound tripterygium wilfordii external application",is a compound external preparation of traditional Chinese medicine developed for the core pathogenesis of active RA with damp-heat stasis.Its clinical effect is remarkable,but its mechanism is not clear.It still needs to be further explored in combination with the latest research progress.Objective1.To determine the changes of metabolic spectrum of articular effusion in RA patients with dampness-heat stasis syndrome.2.To find out the reasons for reducing toxicity and increasing efficiency of Qingre Huayu gel.3.To explore the mechanism of Qingre Huayu gel in the treatment of RA.To clarify the anti-inflammatory and bone-protecting effects and the reduction of key molecules in glucose metabolism reprogramming of Qingre Huayu gel.Methods1.Based on the analysis of the characteristics of metabolic spectrum of articular effusion in patients with RA damp-heat stasis syndrome,19 patients with damp-heat stasis syndrome and 14 patients with non-damp-heat stasis syndrome were included.The method of pseudotargeted metabolomics was used to identify metabolites and analyze metabolic pathway.2.Mass spectrum analysis and transdermal experimentThermo QE plus liquid chromatography tandem high resolution mass spectrometer and Compound discover data processing software were used to test and analyze the non-target chemical composition of Qingre Huayu gel.Carry out in vitro permeation test,including experimental sample preparation,transdermal experimental operation,and transdermal sample analysis,in order to determine the effective transdermal components of Qingre Huayu gel.3.Vitro experimentPreparation of Qingre Huayu gel freeze-dried powder,isolation of rat fibroblastlike synoviocytes(FLS)and monocytes to establish FLS-monocyte co-culture system,respectively set up normal group,model group,HK2 inhibitor group,emodin group,Qingre Huayu gel group.The content of MMP1,MMP13 and HIF-1α in the supernatant of the co-culture system was detected by ELISA,the expression of RANKL and OPG mRNA in the co-culture system was detected by PCR,and the formation of osteoclasts was observed by TRAP staining.4.Vivo experiment6-week-old female rats were selected and 40 were randomly selected to establish CIA model.Normal,model,Iguratimod,emodin and Qingre Huayu gel groups were set up with 10 rats in each group.From the 10th day after the first establishment of the model,the normal group and the model group were given pure water on the knee-ankle joint,the Iguratimod group was given 4.5mg/kg/d,the emodin group was given 40mg/kg/d,and the Qingre Huayu gel group was coated with 0.5g crude drug/kg for 28 days.The general condition of rats in each group was observed daily,the toe swelling volume and the clinical index score of arthritis were measured,and the pathological changes of ankle joint were evaluated by histomorphology.The knee and ankle joints were scanned by micro-CT.Detection of serum HIF-1α,IFN-γ,IL-6 and IL-17 by ELISA.Detection of key molecules of glucose metabolism HK2,SIRT1 and HIF-1αin synovium by Western Blot.The distribution of HIF-1α in knee-ankle joints were observed by immunohistochemical method.Results1.Pseudo-targeted metabolomics showed that compared with non-damp-heat stasis syndrome,the over-expressed metabolites in particular effusion of patients with dampheat stasis syndrome were:PC(20:5(5Z,8Z,11Z,14Z,17Z)/0:0),plasmenyl-PC 38:3,4Acetamidobutanoate,Cis-Aconitic acid,1D-myo-Inositol 1,4,5,6-tetrakisphosphate,4,7,10,13-hexadecatetraenoic acid,3E,8Z,11Z-T etradecatrienyl acetate,1Methylguanosine,Methyl linolenate,Dihomo-γ-Linolenoyl PAF C-16.The low-expressed metabolites were:Indole-3-acetate,Sebacic acid,SM 42:3,Kynurenine,PC(18:2(9Z,12Z)/20:5(5Z,8Z,11Z,14Z,17Z))[U],Uridine 5monophosphate,4-Hydroxy-5-(dihydroxyphenyl)-valeric acid-O-methyl-O-sulphate,5-Hydroxytryptophan,4-Hydroxyquinoline,2-Methyl-1-pyrroline.Metabolic pathways change are mainly related to Glycerophospholipid metabolism,Linoleic acid metabolism,Tryptophan metabolism,alpha-Linolenic acid metabolism,Terpenoid backbone biosynthesis,Tricarboxylic acid cycle,Sphingolipid metabolism,Phosphatidylinositol signaling system,Inositol phosphate metabolism,Glyoxylate and dicarboxylate metabolism,Arachidonic acid metabolism,Arginine and proline metabolism,Pyrimidine metabolism,Tyrosine metabolism.2.Mass spectrometric analysis showed that based on the intersection of UHPLCQ-TOF/MS and TCMSP,33 chemical constituents in Qingre Huayu gel were identified as triptolide,Kaempferol,Fraxetin,Succinic acid,Wilforine,Wilforlide A,Protocatechualdehyde,Rheic acid,Emodin,Citric acid,Aloeemodin,Cinnamic acid,Sennoside A,Chrysophanol 8-O-β-D-glucoside,Senkyunolide A,Caffeic acid,Adenine,Sinapic acid,Phellopterin,Oxypeucedanin,Isoimperatorin,Suberosin,Byakangelicin,Isopimpinellin,Coumarin,Imperatorin,Lupeol,Acetyl-11-keto-βboswellic acid,(-)-Caryophyllene oxide,Eriodictyol,Naringenin,Luteolin,Taxifolin.Only Rhein,the main component of rhubarb,was detected in vitro permeation test.3.In the vitro experiment,MMP1,MMP13 and HIF-α in the model group were significantly higher than those in the normal group(p<0.01),while MMP1,MMP13 and HIF-1 a in each treatment group were significantly lower than those in the model group(p<0.01),suggesting that the inflammatory reaction decreased after treatment.MMP1 and HIF-1α in the Qingre Huayu gel group were significantly lower than those in the emodin group(p<0.05),and MMP13 in the Qingre Huayu gel group was significantly lower than that in the HK2 inhibitor group(p<0.05).In the co-culture system,the expression of RANKL mRNA in the model group was significantly higher than that in the normal group(p<0.05),and the expression of RANKL/OPG in the model group was significantly higher than that in the normal group(p<0.05).There were different degrees of improvement in each treatment group,in which the RANKL mRNA and RANKL/OPG in the Qingre Huayu gel group were significantly lower than those in the model group(p<0.01),and the OPG mRNA was significantly higher than that in the model group(p<0.05).At the same time,RANKL/OPG in Qingre Huayu gel group was significantly lower than that in HK2 inhibitor group(p<0.01).TRAP staining showed that the number of osteoclasts in the model group increased significantly,while the number of osteoclasts decreased in the Qingre Huayu gel group.4.In the vivo experiment4.1 General observationThe rats in the normal group had no special changes,while the rats in each model group showed loss of weight,spirit,appetite and decreased physical strength,which was easy to be irritated.With the increase of treatment days,the overall symptoms of each treatment group were improved compared with the model group in the same period.The weight of each treatment group was lower than that of the normal group,but higher than that of the model group.The joints of rats in the normal group remained normal.The rats in each model group began to show slight swelling of the joint and the skin color became red gradually on the 10th day after immunization,and then the symptoms of joint swelling gradually increased,reaching the peak at about 17-21 days after immunization,and the front paw also showed varying degrees of swelling,at the same time,the joint was dodged when touching,and curled up was limited;27 days after immunization,the joints were deformation and ankylosis.The arthritis index in each treatment group was lower than that in the model group at the same time.The score of each treatment group was lower than that of the model group through the measurement of toe volume meter,it was found that there was no significant change in the volume of both feet in the normal group.From the 10th day of immunization,the joint swelling and displacement of rats in each model group gradually increased,and the volume of both feet increased most obviously from the 17th to 21st day.Then the volume of both feet decreased gradually,but continued to be higher than that of normal rats in the same period,and the degree of joint swelling of rats in each administration group was lower than that in the model group at the same time.4.2 Histomorphology observationHE staining showed that in the normal group,the articular surface was smooth and regular,the articular space was clear,and there was no bone destruction and erosion.In the model group,synovial inflammation was obvious,articular surface was discontinuous and irregular,interstitial fusion disappeared,articular cartilage showed varying degrees of destruction and fibrosis necrotic area and accumulation of a large number of inflammatory cells appeared in soft tissue,accompanied by different degrees of soft tissue edema,synovial tissue abonnal proliferation and filling joint cavity.Slight erosion of articular cartilage could be.seen in Iguratimod group,obvious inflammatory cell infiltration in joint space and discontinuous articular surface in emodin group,and a small amount of inflammatory cell infiltration in joint space in Qingre Huayu gel group.On the whole all the treatment groups were better than the model group.Through micro-CT scan,it was found that the articular surface of the normal group was smooth,the joint space was clear,and there was no bone defect and destruction.Extensive bone erosion and bone destruction appeared on the articular surface of rats in the model group,which were slightly improved in each treatment group,and the bone microstructure parameters showed that the BS/BV ratio of toe to ankle joint in the model group increased significantly(p<0.01)and the destruction of bone surface was serious.The trend of each treatment group was higher than that of the model group,but only Iguratimod reached statistical difference(p<0.05).Qingre Huayu gel group showed an improvement trend.In the model group,Tb.N and Tb.Th of knee joint and tibia were significantly decreased(p<0.01 or p<0.05),while SMI and Tb.Sp were significantly increased(p<0.01),suggesting that osteoporosis was obvious in the model group.Compared with the model group,the SMI of each treatment group decreased,especially lguratimod(p<0.01),followed by Qingre Huayu gel(p<0.05),emodin did not reach statistical difference;Tb.Th of each treatment group is higher than that of the model group,but the statistical difference has not been reached.The Tb.N in each treatment group was significantly higher than that in the model group(p<0.01),Tb.Sp.was significantly lower than that in the model group(p<0.01),suggesting that osteoporosis in each treatment group was significantly better than that in the model group.4.3 The result of exploring the mechanism of actionThe expression levels of HIF-1α,IFN-γ,IL-6 and IL-17 in serum of rats in the model group were significantly higher than those in the normal group(p<0.01).The expression levels of HIF-1 α.IFN-γ,IL-6 and IL-17 in each treatment group were significantly lower than those in the model group(p<0.05 or p<0.01).The protein expression levels of HK2,SIRT1 and HIF-1 a in the knee joint capsule and synovium of the model group were significantly higher than those of the normal group(p<0.01).After treatment,the levels of HK2,SIRT1 and HIF-1α in the Qingre Huayu gel group were significantly lower than those in the model group(p<0.05 or p<0.01).Immunohistochemistry showed that Qingre Huayu gel could effectively reduce the content of HIF-1α in ankle and knee joints.4.4 Safety observationThe pathological changes of liver,kidney and ovary in Qingre Huayu gel group were not significantly different from those in normal group.It is suggested that it is safe and has no obvious side effects.Conclusion1.RA of damp-heat stasis syndrome has metabolic disorder,and the metabolic change of articular effusion are mainly related to 14 abnormal metabolic pathways such as:Glycerophospholipid metabolism,Tricarboxylic acid cycle,Tryptophan metabolism.2.Mass spectrometry analysis and transdermal experiment showed that the toxic components of Qingre Huayu gel decreased,while there were anti-inflammatory components,suggesting that it can effectively inhibit synovitis in the treatment of RA,and reduce adverse reactions.3.The vitro experiment confirmed that Qingre Huayu gel play a therapeutic role in RA.It could effectively reduce the levels of MMP1,MMP13 and HIF1 α in FLSmonocyte co-culture system,down-regulate RANKL/OPG,reduce the number of osteoclasts.4.The vivo experiment confirmed that Qingre Huayu gel could inhibit the glucose metabolism reprogramming by down-regulating the levels of HIF-1α,HK2 and SIRT1,the key molecules of glucose metabolism reprogramming in synovial tissue.It is suggested that Qingre Huayu gel can effectively improve the hypoxia state of synovial microenvironment,inhibit the reprogramming of glucose metabolism,improve the histomorphology of knee joint and ankle joint of CIA rats,play the role of antiinflammation and bone protection.