| ObjectiveBy comparing the changes of serum exosomal micro RNA expression in patients with knee osteoarthritis(KOA)before and after acupuncture treatment,key micro RNAs related to acupuncture effects were screened.Compare the differences between micro RNAs reported in the literature and those found in this study,and conduct in vitro experiments based on the selected key micro RNAs to study their effects on chondrocyte proliferation and apoptosis in order to explore the potential regulatory molecular mechanism of acupuncture for KOA.Methods1.Use the bibliometric visualization software Citespace and VOSviewer to perform literature mining on KOA micro RNA-related research,and select the hot micro RNAs and the main targets in the current research;2.A total of 40 KOA patients and 17 healthy subjects were included,and the KOA patients were randomly divided into acupuncture treatment groups and waiting list.The acupuncture treatment group selected Heding,Neixiyan,Dubi,Xuehai,Liangqiu,Yinlingquan,Yanglingquan,Zusanli for acupuncture treatment every other day,3 times a week,and rest 2 days on weekend,a total of 12 times.The awaiting list did not intervene at the same time as the acupuncture intervention,and the patients were given acupuncture treatment after 4 weeks.All KOA patients were included in the Western Ontario and Mc Master Universities Osteoarthritis Index Visualization Scale(WOMAC)and SF-12 at 0 and 4 weeks after enrollment.The efficacy of acupuncture was evaluated.3.In each group,15 subjects were selected for micro RNA research,of which 10 were subjected to high-throughput sequencing,and 5 were used for later RT-PCR verification experiments.Blood samples of KOA patients were collected at 0 and 4 weeks after enrollment;blood samples of healthy subjects were collected at 0 week of enrollment.After the serum was collected,the exosomes were separated by ultracentrifugation,and the morphology of the separated exosomes was observed under a transmission electron microscope.The expression of CD9,CD63,and CD81 on the surface of exosomes was detected by Western blot.Nanoparticle tracking analysis(NTA)technology to identify exosomes.Exosomal micro RNAs was performed using small RNA sequencing technology.According to the principle of P <0.05,multiples of difference and high expression abundance,differentially expressed micro RNAs were screened.Differential expression profiles of serum exosomes micro RNAs in KOA patients and healthy subjects,differential expression profiles of serum exosomes micro RNAs before and after acupuncture,and the waiting list.The Venny diagram was used to screen out key micro RNAs for acupuncture treatment of KOA.RT-PCR detection of selected micro RNAs to verify sequence accuracy.Target Scan and mi RDB were used to predict target genes for the selected micro RNAs for acupuncture treatment of KOA;GO and KEGG analysis was used to further clarify the related functions and signal pathways of target genes.4.Chondrocytes were selected as the target cells for subsequent research based on the results of literature research,high-throughput sequencing,and RT-PCR verification.After chondrocytes were extracted and cultured,and the cells were identified by immunohistochemistry,immunofluorescence and toluidine blue staining,chondrocytes were screened with the selected micro RNA 338-3p inhibitor and micro RNA 15b-3p mimic,respectively.Transfection,stimulation of chondrocytes with lipopolysaccharide to simulate cell inflammation,detection of cell proliferation with CCK-8 kit,and detection of apoptosis with flow cytometry.Results1.KOA micro RNA research literature mining results: A total of 850 articles were retrieved.The study of mi RNAs related to osteoarthritis began in 2007 and has an increasing trend.The hot micro RNAs in this field are: mi R-140,mi R-146,Mi R-26,mi R-34,mi R-27,mi R-181,mi R-193,mi R-30,mi R-233,mi R-206.2.The effect of acupuncture on KOA patients: After four weeks of treatment in the acupuncture group,the total WOMAC score and WOMAC pain score decreased,and the differences were statistically significant(P < 0.05);WOMAC stiffness score,WOMAC function score,SF-12 PCS,SF-12 MCS did not change significantly,and the difference was not statistically significant(P > 0.05).After waiting for four weeks in the treatment group,there was no significant difference in the total score of WOMAC,WOMAC pain score,WOMAC stiffness score,WOMAC function score,SF-12 PCS,and SF-12 MCS scores(P > 0.05).3.Results of studies on serum exosomal micro RNA expression: Compared with healthy subjects,patients with KOA had a total of 207 differentially expressed serum exosomal micro RNAs,of which 104 were up-regulated and 103 were down-regulated;of these,has-mi R-490-3p was negatively correlated with the womac pain score;has-mi R-1255 b was positively correlated with the womac pain score.In the acupuncture group,there were 65 differentially expressed exosome micro RNAs compared with those before treatment,of which 32 were up-regulated and 33 were down-regulated.In waiting list,there were 76 differentially expressed micro RNAs,of which 43 were up-regulated and 33 were down-regulated.4.The mi RNAs down-regulated by acupuncture for KOA were: hsa-mi R-504-3p,hsa-mi R-1915-3p,hsa-mi R-103a-2-5p,hsa-mi R-887-3p,hsa-mi R-1228-5p,hsa-mi R-34c-3p,hsa-mi R-3168,hsa-mi R-518e-3p,hsa-mi R-1296-5p,hsa-mi R-338-3p,hsa-mi R-199b-5p;the mi RNAs up-regulated in KOA with acupuncture were: hsa-mi R-514a-3p,hsa-mi R-4440,hsa-let-7f-5p,hsa-mi R-15b-3p.Among them,mi R-338-3p and mi R-15b-3p are mi RNAs reported in the literature.5.RT-PCR verification results: The expression of hsa-338-3p was increased in KOA patients(P = 0.332),and decreased after acupuncture treatment(P = 0.149),but there was no significant change after the waiting observation period(P = 0.647),but the differences were not statistically significant(P > 0.05).The expression of hsa-199b-5p was increased in patients with KOA(P = 0.305),and it still showed an increase after acupuncture treatment(P = 0.252).There was no significant change after the waiting observation period(P = 0.536).The expression of has-mi R-15b-3p was increased in patients with KOA(P = 0.003),the difference was statistically significant;after acupuncture and waiting for observation period,they were slightly reduced(P = 0.661,P = 0.083),but the difference was not statistically significant(P > 0.05).The expression of hsa-mi R-1296-5p was increased in patients with KOA(P < 0.001),and the difference was statistically significant;both decreased after acupuncture and waiting for observation period(P = 0.015,P = 0.024),The difference was statistically significant.hsa-mi R-3168 was not significantly different between KOA patients and healthy subjects(P = 0.923),and increased slightly after acupuncture and waiting for observation period(P = 0.881,P = 0.1736).6.Bioinformatics results: predictions of mi R-338-3p,mi R-15b-3p,mi R-199b-5p,mi R-1296-5p target genes predicted that the intersection of mi RDB and Targetscan predicted a total of 262 target genes.KEGG pathyway enrichment analysis of a total of 37 pathways,mainly involving cancer,apoptosis,B cell receptors,cholinergic synapses,dopaminergic synapses,and other biochemical metabolic pathways,as well as MAPK,TNF,m TOR,PI3K-Akt,Wnt,B cell receptor,HIF-1,AMPK,Erb B and other pathways.GO enrichment analysis of 262 predicted target genes is mainly involved in regulating RNA metabolism,DNA template,nuclease complex metabolism,nucleic acid template transcription,and RNA biosynthesis.7.The proliferation of mi R-338-3p inhibitor cells was significantly increased after transfection,the difference was statistically significant(P < 0.001);the apoptosis rate of mi R-338-3p inhibitor was slightly reduced after transfection,and the difference was not statistically significant(P > 0.05).After mi R-15b-3p mimic transfection,cell proliferation increased slightly,the difference was not statistically significant(P < 0.001);after mi R-15b-3p mimic transfection,the apoptosis rate decreased,and the difference was statistically significant(P < 0.01).Conclusions1.Literature research on KOA mi RNA found that chondrocytes are the core of this field,which mainly involves mechanisms such as cell proliferation,apoptosis and cartilage formation.2.Clinical studies showed that KOA patients could effectively relieve joint pain in KOA patients after acupuncture treatment.3.Sequencing of serum exosomes in KOA patients and healthy people revealed that there were differences in the expression profiles of serum exosomes in KOA patients and healthy people,of which 104 were up-regulated and 103 were down-regulated.Acupuncture treatment could modulate the expression of serum exosome mi RNA in KOA patients,of which mi R-338-3p,mi R-15b-3p,mi R-199b-5p,mi R-3168,mi R-1296-5p might be the Key mi RNA in acupuncture treatment of KOA.Further bioinformatics analysis found that acupuncture might play a therapeutic role by affecting MAPK pathway and apoptosis.4.Verification through in vitro cytology experiments revealed that mi R-338-3p could promote chondrocyte proliferation,and mi R-15b-3p could inhibit chondrocyte apoptosis,which suggesting that this might be one of the mechanisms of acupuncture treatment of KOA. |
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