Clinical And Mechanism Study Of Duhuojisheng Decoction Regulating MiR-223 Mediated JAK/STAT Signaling Pathway In Treatment Of Lumbar Disc Herniation

Objects:Through clinical observation of the difference between the Duhuojisheng Decoction and sustained-release tablets in the treatment of lumbar disc herniation,the clinical efficacy of the treatment of lumbar disc herniation was clarified;the paper explored the pathogenesis of intervertebral disc degeneration,clarified the correlation of intervertebral disc degeneration and miR-223/JAK/STAT pathway,and further explored the mechanism of miR-223 regulation of JAK/STAT signal pathway to slow lumbar disc degeneration.Methods:The first part of clinical research: A retrospective analysis was performed on 162 patients with lumbar disc herniation treated in the orthopedic outpatient department of our hospital from September 2020 to September 2022.According to the case selection criteria of this study,they were divided into Duhuojisheng Decoction group(group A)and etontoacid sustained-release tablet group(group B),including 77 cases in group A and 85 cases in group B.VAS score,JOA score and Oswestry Disability Index scores were observed before treatment,1 week after treatment,2 weeks after treatment,and 4 weeks after treatment to evaluate the clinical efficacy and safety of Duhuojisheng Decoction.The second part: In this study,clinical samples of human lumbar disc degraded nucleus pulposus tissue and non-degraded nucleus pulposus tissue were taken as the research object.(1)The tissue specimens obtained during the operation were placed in a50 ml aseptic centrifuge tube containing 20 ml DMEM high-glucose culture medium,stored at low temperature and sent to the laboratory within 30 min for cell isolation,extraction and culture of nucleus pulposi cells,and cell identification by immunofluorescence technology.(2)The expressions of miR-223 and JAK/STAT in degraded and non-degraded nucleus pulposus tissue samples were detected by qPCR.The third part: In this part,we will study the mechanism of miR-223 regulation of JAK/STAT signaling pathway in nucleus pulposus cells.(1)nucleus pulposus cells of lumbar intervertebral disc were taken as the object of study.Nucleus pulposus cells extracted from the second part were resuscated and passed to the second generation.Five groups were set up,respectively blank group,Mimic control transfection group,miR-223 mimics transfection group,Inhibitor control transfection group.miR-223 Inhibitor transfection group.After transfection,the m RNA expression levels of STAT factors were detected by qPCR and p-JAK protein expression levels were detected by Western Blot.(2)According to the above experimental results,a lentiviral vector of STATx was constructed to observe the specific mechanism of miR-223 regulation of JAK/STAT signaling pathway in NP cells in an LPs-induced inflammatory environment.WT NP cells,control virus NP cells and STATx down-regulation NP cells were set.Six groups were set for each cell,which were blank group,LPS+mimic control transfection group,and LPS+mimic control transfection group.LPS+miR-223 mimics transfection group,LPS+inhibitor control transfection group,miR-223 inhibitor transfection group.NP cells were treated with10μg/m L LPS.qPCR was used to detect gene expression level,Western Blot was used to detect protein expression level,ELISA was used to detect inflammatory level,flow cytometry and Tunel staining were used to detect cell apoptosis.To focus on the mechanism of miR-223 intervention in JAK/STAT pathway regulating apoptosis,expression of inflammatory factors and stroma-degrading enzymes,and extracellular matrix synthesis in LPs-induced nucleus pulposus cells.The fourth part: In this part,NP cells of lumbar intervertebral disc were studied.To study the changes of miR-223/JAK/STAT pathway and downstream factors after the intervention of Duhuojisheng Decoction in nucleus pulposus cells,and to clarify the mechanism of action of Duhuojisheng Decoction in nucleus pulposus cells of interdisc.(1)To prepare the medicated serum of Duhuojisheng Decoction and establish the model of inflammatory NP cells induced by LPS.The study was divided into four groups: blank group(LPS+ control serum),low dose group(LPS+2%DXJS medicated serum +8% fetal bovine serum),medium dose group(LPS+5%DXJS medicated serum +5% fetal bovine serum)and high dose group(LPS+10%DXJS medicated serum).The proliferation of NP cells in each group was detected by CCK-8 method,and the apoptosis of NP cells in each group was detected by flow cytometry and Tunel staining.(2)The optimal concentration of medicated serum was selected to transfect the cells,and LPS+DXJS+mimic control transfection group and LPS+DXJS+miR-223 mimic transfection group were set.The proliferation of NP cells after transfection was detected by CCK-8 method,and the apoptosis of NP cells after transfection was detected by flow cytometry and Tunel staining.qPCR was used to detect gene expression level,and Western Blot was used to detect protein expression level.Results:The first part of clinical research:(1)In terms of VAS score,there was no significant difference between the two groups before treatment(P=0.84),and there were statistical differences in VAS scores between the two groups at 1,2 and 4 weeks after treatment(P<0.05),as shown in Table 1-3.The frequency of VAS scores before and after treatment changed from large to small.The overall trend of VAS scores before and after treatment between the two groups was decreased from high to low.(2)In terms of JOA score,there was no significant difference between the two groups before treatment(P=0.61),and there were statistical differences between the two groups at 1,2 and 4 weeks after treatment(P<0.05).The frequency of each JOA score before and after treatment changed from small to large.The overall trend of JOA scores before and after treatment was increased from low to high.(3)In terms of ODI,there was no significant difference in ODI scores between the two groups before treatment(P=0.50),and there were statistical differences in ODI scores between the two groups at 1,2 and 4 weeks after treatment(P<0.05).The frequency of ODI scores before and after treatment changed from large to small.The overall trend of ODI scores before and after treatment between the two groups was decreased from high to low.(4)Four weeks after treatment,the effective rate of group A was 74.03%,and the total effective rate of group B was 65.88%.The second part:(1)Immunofluorescence detection showed the presence of polyproteoglycan and Collagen II in the cells,which proved to be NP cells.(2)The expression of miR-223 in the nucleus pulposus of degenerative intervertebral discs was significantly higher than that in the nucleus pulposus of non-degenerative intervertebral discs,and there was a significant difference between the two(P < 0.01).This difference in expression level suggests that the intervertebral disc degeneration is related to miR-223.(3)m RNA expressions of Jak2(P < 0.01),Jak3(P<0.01),stat1(P < 0.01),stat4(P < 0.01)and stat5B(P < 0.05)in degraded nucleus pulposus tissues were significantly increased compared with those in undegraded nucleus pulposus tissues.The m RNA expressions of stat3(P < 0.05)and stat5A(P < 0.01)were significantly decreased.There was no significant difference in the m RNA expression of Jak1,stat6,and stat2.Similarly,this difference indicated that Jak2,Jak3,stat1,stat4,and stat5 B were associated with intervertebral disc degeneration.The third part:(1)After NP cells transfected with miR-223 mimics detected by qPCR,STAT1 m RNA expression was increased compared with blank group and Mimic control transfection group.After NP cells transfected with miR-223 Inhibitor,Compared with blank group and Inhibitor control transfection group,m RNA expressions of STAT1 and STAT6 were increased,and the increase of STAT1 was larger than that of STAT6.Therefore,according to the qPCR results,the STAT1 sh RNA virus and control virus were packaged for follow-up experiments.(2)Mir-223-mimics significantly increased the expression of JAK2 and STAT1,and miR-223 inhibitors significantly decreased the expression of JAK2.This proves that miR-223 mainly up-regulates the expression of JAK2/STAT1 in NP cells.(3)WB test results showed the expression of caspase-1 and caspase-3 in each sample: In control virus NP cells and WT NP cells,the expression levels of caspase-1 and caspase-3 were higher than those in the blank group after LPS treatment,indicating that LPS can stimulate the expression of caspase-1 and caspase-3 in these two cells.After the combined action of LPS and miR-223 mimics on these two cells,the expression levels of caspase-1 and caspase-3 were higher than those of LPS group,indicating that miR-223 mimics can stimulate the expression of caspase-1 and caspase-3 in these two cells.After the combined action of LPS and miR-223 Inhibitor on caspase-1 in the two types of cells,the expression of caspase-3 was lower than that in the LPS group,indicating that miR-223 Inhibitor can inhibit caspase-1 and caspase-3 expression in these two types of cells.In STATx down-regulation NP cells,the expression levels of caspase-1and caspase-3 in each group had no significant changes compared with the blank group.(4)In control virus NP cells and WT NP cells,the proportion of apoptotic cells after LPS treatment was higher than that in blank group.The fourth part:(1)The results of CCK-8 method showed that the higher the concentration of Duhuojisheng Decoction,the stronger the activity of NP cells,the stronger the proliferation ability;Flow cytometry showed that the higher the concentration of Duhuojisheng Decoction,the lower the level of apoptosis of NP cells.(2)After transfection,CCK-8 assay showed that miR-223 reduced the activity and proliferation of NP cells.Flow cytometry showed that miR-223 increased the apoptosis level of NP cells.(3)After transfection,the qPCR results showed that the expression of miR-223 was significantly increased in the transfection group with LPS+DXJS+mimic control(P < 0.01).The m RNA expressions of Caspase-1(P < 0.01),Caspase-3(P < 0.01),MMP3(P < 0.01),MMP13(P < 0.01),IL-6(P < 0.01)and TNF-α(P < 0.01)were significantly increased.(4)After transfection,WB results showed that compared with the transfection group with LPS+DXJS+mimic control,The expressions of P-JAK2,p-Stat1,Caspase-1,Caspase-3,MMP3,MMP13,IL-6 and TNF-α were significantly increased after transfection with LPS+DXJS+miR-223 mimic(P < 0.01).Conclusions:(1)Duhuojisheng Decoction can significantly relieve pain,improve dysfunction and improve quality of life in patients with lumbar disc herniation.Compared with etoduc sustained release tablets,the curative effect is more reliable.(2)miR-223/JAK2/STAT1 pathway plays an important regulatory role in the pathological progression of lumbar disc degeneration,and is an important pathway for the pathogenesis and prevention of lumbar disc degeneration.(3)Duhuojisheng Decoction can inhibit miR-223/JAK2/STAT1 pathway to reduce the inflammatory response of nucleus pulposus cells and slow down the excessive apoptosis of nucleus pulposus cells of lumbar disc.