| Objective:Rheumatoid arthritis(RA)is an autoimmune disease with synovitis as the pathological basis and erosive arthritis as the main feature.The etiology of RA is unknown and can’t be cured yet.Traditional Chinese medicine has the advantages of definite curative effect,small side effects,treating both manifestation and root cause of disease,as well as increasing efficacy and reducing toxicity when used with western medicine.Si Xian Zhu Bi Decoction is professor Peng Rui’s empirical prescription for clinical treatment of RA under the guidance of the theory of "Yangyuan Tongluo".This subject intends to explore the theoretical basis of Si Xian Zhu Bi Decoction in treating RA,observe the influence of Si Xian Zhu Bi Decoction on CIA rats,screen the potential miRNAs regulatory factors and regulatory pathways of Si Xian Zhu Bi Decoction in treating RA based on network pharmacology and metabolomics technology,and study the effect of Si Xian Zhu Bi Decoction on the miR-17-5p/PI3K/AKT/FASN axis in the synovium of CIA rats and MH7A cells,and further study the mechanism of Si Xian Zhu Bi decoction mediated MH7A cell apoptosis through miR-17-5p/PI3K/AKT/FASN axis,providing experimental basis for the treatment of RA with Si Xian Zhu Bi decoction.Methods:1.This part discussed the pathogenesis of RA,the understanding of TCM on RA,the relationship between the theory of "Yangyuan Tongluo" and RA,and the analysis of the prescription of Si Xian Zhu Bi Decoction in the treatment of RA,in order to explain the theoretical basis of Si Xian Zhu Bi Decoction in the treatment of RA.2.The CIA rat model was established by injecting bovine type II collagen emulsifier into the tail root of SD rats.The successful CIA rats were randomly divided into Model group(Model),high,medium and low dose groups of Si Xian Zhu Bi Decoction(SXZBTH,SXZBTM,SXZBTL),and methotrexate group(MTX),with 8 rats in each group.8 rats without modeling at the same experimental stage were randomly selected as Control group.The rats in SXZBTH group,SXZBTM group and SXZBTL group were given Si Xian Zhu Bi Decoction at the dose of 30.24g/kg/d,15.12g/kg/d and 7.56g/kg/d respectively according to their body weight;Rats in the MTX group were gavaged with 1.05mg/kg MTX suspension twice a week;Rats in the Control group and Model group were gavaged with 10 m L/kg/d of normal saline.Continuous intervention for 6 weeks.The behavioral changes of rats were observed.Arthritis index(AI)and hind paw volume were used to evaluate the severity of arthritis,HE staining was used to observe the pathological changes of joints,liver and spleen,and ELISA was used to detect the levels of IL-6,TNF-αand IL-17 in serum of rats in each group.3.The method of network pharmacology was used to preliminarily predict the active components,process and pathways of Si Xian Zhu Bi Decoction in the treatment of RA.The difference of serum metabolism of CIA rats before and after treatment with Si Xian Zhu Bi Decoction was analyzed by using non-targeted Metabolomics based on LC-MS,to screen the potential biomarkers and metabolic pathways of Si Xian Zhu Bi Decoction in the treatment of RA.Integrate the results of network pharmacology and metabolomics to screen target genes.Find the relevant miRNAs between the target gene that involved in RA through the database.Collect and sort out the literatures,analyze and predict the functional pathway of Si Xian Zhu Bi Decoction in treating RA.4.The synovial tissues of rats in Control,Model,SXZBTH,SXZBTM,SXZBTL,MTX group were extracted.RT-qPCR method was used to detect miR-17-5p,PI3K,AKT,FASN mRNA expressions in synovial tissues of rats in each group.WB method was used to detect the expression level of PI3K,p-AKT,FASN,Bax,Bcl-2,C-Caspase3,C-Caspase9 protein in synovial tissue of rats in each group.5.The MH7A cell line was selected as the in vitro model of RA,and the MH7A cell inflammation model was induced by LPS stimulation.MH7A cells were set as blank group,and MH7A cells induced by LPS were set as Control group.LPS-induced MH7A cells interfering with 10% and 20% Si Xian Zhu Bi Decoction medicated serum were divided into 10% drug-contained group and 20%drug-contained group respectively.RT-qPCR was used to detect the expression of miR-17-5p in the cells of each group,CCK8 and Annexin V-PI flow cytometry were used to detect the trend of cell proliferation and apoptosis in each group,and WB was used to detect the expression level of PI3K,p-AKT,FASN,Bax,Bcl2,C-Caspase3,C-Caspase9 protein in the cells of each group.6.The LPS-induced MH7A cells transfected with miR-NC,miR-17 mimics,miR-inhibitor-NC,and miR-17 inhibitor were recorded as miR-NC group,miR-17 mimics group,anti-miR-NC group,and anti-miR-17 group;The LPS-induced MH7A cells transfected with miR-inhibitor-NC and miR-17 inhibitor-NC were treated with 20%medicated serum,which were recorded as SXZB+anti-miR-NC group and SXZB+anti-miR-17 group respectively.RT-qPCR was used to detect the expression of miR-17-5p in the cells of each group,flow cytometry were used to detect the trend of cell apoptosis in each group,and WB was used to detect the expression level of PI3K,p-AKT,FASN,Bax,Bcl2,C-Caspase3,C-Caspase9 protein in the cells of each group.Results:1.According to the theory of traditional Chinese medicine’s vital energy,the deficiency of vital energy is the main pathogenic factor for RA.Evil factors such as wind,cold,and dampness invade the muscle surface,and the disharmony of qi and blood,as well as the blockage of meridians and collaterals,are the key to its onset.Si Xian Zhu Bi Decoction attributs to the lung,spleen,liver and kidney channel,playing the role of nourishing vital energy and relieving obstruction and unblocking collaterals.The effective ingredients in the prescription,such as Curculigo,Epimedii,Clematis,hairyvein agrimony,Radix Paeoniae Alba,Glycyrrhizae,Scorpio and Centipede,can regulate multiple signal pathways,inhibit the release of inflammatory factors,reduce inflammatory reaction and joint damage,and achieve the anti-rheumatic effect.2.After 8 weeks of modeling,rats’ ankle joints in the Model group were obviously swelling and hyperemia,and some rats had joint deformity.Swelling and hyperemia of ankle joint of rats in SXZBTL,SXZBTM,SXZBTH and MTX groups were alleviated to varying degrees,and the reduction of joint swelling in SXZBTH group rats was the most obvious.From the 14th day,compared with the Control group,the AI index of rats in the Model group increased significantly(P<0.05).Since the 28 th day,the AI index of the four groups after drug intervention was lower than that of the Model group(P<0.05).From the 35 th day,compared with the Model group at the same experiment stage,the toe volume of rats in the other four groups after drug intervention gradually decreased(P<0.05).Compared with their own group on the 14 th day of modeling,the AI index of the four groups of rats after drug intervention on the 56 th day decreased significantly(P<0.05),and the toe volume decreased gradually(P<0.05).The results of ELISA showed that compared with the Control group,the contents of TNF-α,IL-6 and IL-17 in the serum of rats in the Model group were significantly higher(P<0.05).Compared with Model group,the contents of TNF-α,IL-6 and IL-17 in the serum of rats in SXZBTM group and SXZBTH group were significantly decreased(P<0.05).Compared with the Control group,the liver index of MTX group was significantly higher(P<0.05),while that of Model group was significantly lower(P<0.05).There was no significant difference in spleen index of rats in each group(P>0.05).The HE staining section of liver showed that the hepatocytes of rats in each group were arranged in a basic rule,without deformation and necrosis.There are a few red blood cells in the central vein of hepatic lobule of rats in Model group,MTX group and SXZBTL group.In Model group,the hepatic cord space was slightly widened,while in MTX group,there were more red blood cells in hepatic sinuses.HE staining section of spleen showed that the spleen tissue structure of rats in each group was basically normal.From the HE pathological section of the ankle joint,it can be seen that the cartilage surface of the ankle joint in the Control group is smooth,the synovial tissue is smooth and transparent,without hyperemia and hypertrophy,and no obvious inflammatory cell infiltration.But in the Model group,the cartilage surface of rats was rough,partially defective,the joint cavity was narrow,deformed or even occluded,the synovial tissue hyperplasia was obvious,and eroded to the cartilage surface,the synovial cells arranged in disorder,and a large number of inflammatory cells infiltrated.The pathological signs of ankle joint in SXZBTL group,SXZBTM group,SXZBTH group and MTX group were less severe than those in Model group.Compared with MTX group,the cartilage surface of SXZBTM group and SXZBTH group was smoother and the proliferation of synovial tissue was lighter.3.The results of network pharmacology show that the most important core components of Si Xian Zhu Bi Decoction in treating RA include quercetin,kaempferol,luteolin and other active components.GO enrichment analysis shows that Si Xian Zhu Bi Decoction mainly participates in many biological processes such as cellular stress reaction,lipid regulation,inflammatory reaction and so on in the treatment of RA.KEGG enrichment analysis showed that PI3K/AKT signal pathway involved the most common target.The results of serum metabolomics showed that,before and after treatment with Si Xian Zhu Bi Decoction,there were significant differences in19 endogenous metabolites in serum of CIA rats,such as fluoroprostaglandin,palmitic amide,stearic acid,cholic acid,serine,tyrosine,cysteine,prostaglandin,etc.These metabolites mainly involved in bile acid biosynthesis,fatty acid biosynthesis,tyrosine metabolism,etc.Combined with the research results of network pharmacology and metabolomics,we found that FASN is the common target gene of the two research results.Through database and literature analysis,miR-17-5p was found to be the most conservative regulatory factor associated with FASN and involved in the course of RA.4.The results of RT-qPCR showed that compared with the Control group,the expression of miR-17-5p in the synovial tissue of the Model group was significantly lower(P<0.05),and the expression of PI3K,AKT,FASN mRNA was significantly higher(P<0.05);Compared with Model group,the expression of miR-17-5p in synovial tissue of SXZBTL,SXZBTM and SXZBTH groups was significantly higher(P<0.05),and the expression of PI3K,AKT and FASN mRNA was significantly lower(P<0.05).Compared with MTX group,the expression of miR-17-5p in synovial tissue of SXZBTM and SXZBTH group was significantly higher(P<0.05),and the expression of FASN mRNA was significantly lower(P<0.05).The expression of PI3K and AKT mRNA in synovial tissue of SXZBTH group was lower than that of MTX group(P<0.05).WB results showed that the relative expression of Bax,C-Caspase3,C-Caspase9 protein in synovial tissue of rats in Model group was significantly lower than that in Control group(P<0.05),while the relative expression of Bcl-2,PI3K,p-AKT,FASN protein was significantly higher(P<0.05).Compared with the Model group,the relative expression of Bax,C-Caspase3 and C-Caspase9 proteins in the synovium of rats in SXZBTL group,SXZBTM group and SXZBTH group increased significantly(P<0.05),while the relative expression of Bcl-2,PI3K,p-AKT and FASN proteins decreased significantly(P<0.05).Compared with MTX group,the relative expression of Bax,C-Caspase3 and C-Caspase9 protein in synovial tissue of rats in SXZBTL group,SXZBTM group and SXZBTH group increased significantly(P<0.05),and the relative expression of PI3K and p-AKT protein decreased significantly(P<0.05).The relative expression of Bcl-2 protein in SXZBTM group and SXZBTH group was lower than that in MTX group(P<0.05).5.CCK-8 results showed that after 12 h,24h and 48 h of intervention with Si Xian Zhu Bi decoction containing serum,compared with the Control group,the inhibition rate of MH7A cell proliferation in 10% and 20% containing serum gradually decreased(P<0.05),and it showed a dose-dependent increase.The results of flow cytometry showed that the proportion of apoptosis in the cell model increased significantly after 48 hours of intervention with Si Xian Zhu Bi Decoction containing serum(P<0.05).RT-qPCR results showed that the expression of miR-17-5p in MH7A cells in the Control group was significantly lower than that in the blank group(P<0.05);Compared with the Control group,the expression of miR-17-5p in MH7A cells in the 10% drug-contained group and the 20% drug-contained group increased significantly(P<0.05).The WB results showed that compared with the blank group,the relative expression of Bax,C-Caspase3,C-Caspase9 protein in the Control group was significantly lower(P<0.05),and the relative expression of Bcl-2,PI3K,p-AKT,FASN protein was significantly higher(P<0.05).Compared with the Control group,the relative expression of Bax,C-Caspase3,C-Caspase9 protein in the cells of the 10% drug-contained group and the 20%drug-contained group was significantly higher(P<0.05),while the relative expression of Bcl-2,PI3K,p-AKT,FASN protein was significantly lower(P<0.05).6.After 48 hours of transfection,the RT-qPCR results showed that compared with the miR-NC group,the expression level of miR-17-5p in MH7A cells of the miR-17 group was significantly increased(P<0.05),while the expression level in the in-miR-17 group was significantly decreased(P<0.05).The WB results showed that compared with the miR-NC group,the expression of PI3K,p-AKT,FASN,and Bcl2 protein in MH7A cells of the miR-17 group was significantly decreased(P<0.05),while the expression of Bax,C-caspase3,and C-caspase9 protein was increased(P<0.05).In the in-miR-17 group,the expression of PI3K,p-AKT,FASN,and Bcl2 protein in MH7A cells was significantly increased(P<0.05),while the expression of Bax,C-caspase3,and C-caspase9 protein was decreased(P<0.05).The flow cytometry results showed that compared with the miR-NC group,the proportion of apoptotic cells in MH7A cells of the miR-17 group was increased(P<0.05),while in the in-miR-17 group,the proportion of apoptotic cells was decreased(P<0.05).After intervention with Si Xian Zhu Bi Decoction-containing serum for 48 hours,the RT-qPCR results showed that compared with the SXZB+in-miR-NC group,the expression level of miR-17-5p in MH7A cells of the SXZB+in-miR-17 group was significantly decreased(P<0.05).The WB results showed that compared with the SXZB+in-miR-NC group,the expression of PI3K,p-AKT,FASN,and Bcl2 protein in MH7A cells of the SXZB+in-miR-17 group was significantly increased(P<0.05),while the expression of Bax,C-caspase3,and C-caspase9 was decreased(P<0.05).The flow cytometry results showed that compared with the SXZB+in-miR-NC group,the proportion of apoptotic cells in MH7A cells of the SXZB+in-miR-17 group was decreased(P<0.05).Conclusion:1.The treatment principle under the guidance of the "Yangyuan Tongluo" theory is consistent with the pathogenesis of RA.The structure of the Si Xian Zhu Bi decoction and its therapeutic effect conform to the theory of TCM treatment of RA based on the disease mechanism and the "prescription-syndrome correspondence" principle.The components of Si Xian Zhu Bi decoction also have a modern pharmacological research basis,studying its mechanism of action on RA is of great significance.2.Si Xian Zhu Bi decoction can effectively alleviate joint swelling and synovial inflammation in CIA rats,inhibit excessive proliferation of synovium,reduce joint damage,and lower liver damage.The efficacy and safety of Si Xian Zhu Bi decoction are superior to methotrexate.3.Si Xian Zhu Bi decoction mainly exerts its therapeutic effect on RA through active ingredients such as quercetin,naringenin,and hesperidin.Its mechanism of action is related to the regulation of multiple signal pathways such as PI3K/AKT,and participates in the regulation of various biological processes such as cellular stress response,lipid regulation,and inflammatory response.Si Xian Zhu Bi decoction is involved in the bile acid biosynthesis,fatty acid biosynthesis,and tyrosine metabolism pathways in CIA rats.4.Si Xian Zhu Bi decoction can improve joint synovial inflammation in CIA rats,and its mechanism may be related to upregulating the expression of miR-17-5p in CIA rat synovial tissue and MH7A cells,inhibiting the activation of PI3K/AKT/FASN pathway,inhibiting the proliferation of MH7A cells,and promoting it’s apoptosis. |
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