| The first part:miR-328 may regulate the proliferation and apoptosis of NP cells through targeting PAK6The research backgroundDisc degeneration is one of the main causes of low back pain.It not only brings problems to patients' lives,but also imposes a great burden on the public health system.According to incomplete statistics,about 40 percent of people under the age of 30 have symptoms of disc degeneration,while 90 percent of people over the age of 50 do.At present,the pathogenesis of intervertebral disc degeneration is not clear,and there are many theories about its pathogenesis in the scientific community.Ask from anatomical physiology Angle,vertebral plate mainly divided into three layers,the outer structure is mainly composed of dense fibrous ring(annulus fibrosus,AF),middle is the Nucleus pulposus of jelly form(the Nucleus pulposus,NP)organization,inner layer is close to the vertebral body the endplate cartilage.The current mainstream theories think that the main cause of disc degeneration gradually,ask is due to vertebral plate up and down the endplate calcification,caused the nutrients of obstacles,lead to nutritional deficiencies,nucleus pulposus cells produced abnormal apoptosis,which leads to the occurrence of the disease.Therefore,by studying the related mechanism of degenerative nucleus pulposus cells,and select reliable specific target,can be better for intervertebral disc degeneration in the basic research and clinical treatment to provide more theoretical basis and new treatment method.In recent years,a large number of studies have been conducted on the role of micro-ma(miRNAs)in various diseases.MiRNAs is a kind of small non-coding RNA molecules known collectively,its length is about 20?22 nucleotides,and mRNA by transcription coding protein,on the other hand,the effect of the role of micrornas mainly by cutting or targeted directly instead of 3 '-complementary mRNA non-coding region,and the expression of target genes were negative.There is increasing evidence that miRNAs may be involved in the occurrence and development of many diseases,such as cardiovascular diseases,cancer,autoimmune diseases,etc.The role of miRNAs in bone and cartilage development and orthopedic diseases such as osteoarthritis and osteosarcoma has also been discussed.In the field of intervertebral disc degeneration,the role of miRNA has been reported.In a new study,researchers compared patients with intervertebral disc degeneration by using the method of sequencing the miRNA expression in patients with intervertebral disc tissue and normal tissue,found that miR-328 in the tissue of intervertebral disc degeneration expression abnormal increase,however,the related mechanism is still unclear.P-21 protein kinase 6(p21-activated kinases,PAK6)is a serine/threonine protein kinase,in previous studies,research the regulation of hair to its participation in a variety of cell biological processes,such as cell proliferation and apoptosis,rearrangement of the cytoskeleton,NADPH oxidase activity of regulation,silk column activated protein kinase signaling pathway activation,etc.The current research about PAK6 more focused on the tumor area,unusually high expression in PAK6 was found in a wide variety of tumor,including prostate cancer,liver cancer,endometrial adenocarcinoma non-small cell lung cancer,colon cancer,etc.,the study found that inhibit PAK6 can promote the apoptosis of cancer cells,inhibit the proliferation,and can effectively improve the sensitivity of cancer cells to chemotherapy drugs,and the expression PAK6 can promote cell proliferation,inhibit its apoptosis.Through bioinformatics,PAK6 was predicted to be a direct target of mir-328,while the role of PAK6 in intervertebral disc degeneration has not been reported.Therefore,in this study,we will preliminarily explore the role of mir-328 and its target gene PAK6 in the pathogenesis of intervertebral disc degeneration.We compared the expression levels of mir-328 and PAK6 in normal nucleus pulposus cells and nucleus pulposus cells in rats with disc degeneration by establishing a model of intervertebral disc degeneration in rats.In addition,we also will be the original generation of cultivating normal intervertebral disc nucleus pulposus cells of rats,and to the transfection of miR-328 analog and inhibitor,in order to make clear whether the miR-328 through targeted PAK6 control rat intervertebral disc nucleus pulposus cells proliferation apoptosis mechanisms,provide new theoretical basis for the treatment of intervertebral disc degeneration and specific targets.Objective:The pathogenesis of microRNAs involved in intervertebral disc degeneration(IDD)has been discussed,but the role of mir-328 in IDD remains to be further studied.Experimental method:1)model establishment of experimental animals with intervertebral disc degenerationHealthy Wista rats were selected and the intervertebral disc degeneration model of rats was established by puncture according to the previous literature.The specific steps are as follows:Will first anesthetic rats(intraperitoneal injection of 3%sodium pentobarbital,the measurement of 40 mg/kg),after being successful anesthesia,to fix their limbs,prone on the table,using Faxitzon instrument in the X-ray imaging in rats in order to make clear its lumbar segment,according to the imaging result,after selecting the rat 4-5 lumbar,determine the location,with 20 ml syringe needle lumbar puncture of the,by the method of mechanical damage,caused the degeneration of intervertebral disc tissue.Two weeks later,MRI was used to determine the degree of degeneration of intervertebral disc in experimental rats.2)separation and culture of primary degenerative nucleus pulposus cellsAfter establishing the successful model of intervertebral disc degeneration,the model mouse was anesthetized,fixed limbs and prostrate in the operating table.After the skin was cut open,the lumbar spine was cut off,the cells were separated,and the monocyte suspension was made after culture medium was added.After that,the cells were purified by differential adherent culture to obtain the original degenerative nucleus pulposus cells.After successful primary culture,the cells were stained with hematoxylin and eosin,and morphological identification of nucleus pulposus cells was carried out by observing cell morphology and aggregation.3)fluorescence quantitative real-time PCR method was used to detect the expression of mir-328 and related markers of disc degeneration in rat intervertebral disc tissue nucleus pulposus cellsCollect primary separation of degenerative nucleus pulposus cells,and the generation of cultivating normal nucleus pulposus cells,using Trizol reagent,according to the instruction method of extracting total RNA in cells,through reverse transcription to RNA into cDNA,after using the fluorescent quantitative PCR method,compare the degeneration and degenerative nucleus pulposus cells of type ? collagen,collagen type ?tumor-specific markers,type ? collagen,and target of micrornas miR-328mRNA expression levels.4)the expression of intervertebral disc degeneration related markers in nucleus pulposus cells of intervertebral disc tissues in rats was detected by protein immunoblottingCollect the original generation of degenerative nucleus pulposus cells,and the generation of cultivating normal nucleus pulposus cells,using the reagent kit in accordance with the manual method of extraction of total protein,the protein immunoblot method is adopted,after comparing degeneration and tumor-specific markers not degenerative nucleus pulposus cells of type ? collagen,collagen type ?,the expression of type ?collagen protein levels.5)the effect of mir-328 on the proliferation of nucleus pulposus cells was detected by MTT assayThe original generation of cultivating normal nucleus pulposus cells,using liposome analog and inhibitor of miR-328 transfection into the nucleus pulposus cells,culture after 72 hours,determined by MTT method is used to compare transfection group,miR-328 analogue transfection group,and miR-328 inhibitor transfection group of cell proliferation.6)the effect of mir-328 on the apoptosis of nucleus pulposus cells was detected by flow cytometryThe original generation of cultivating normal nucleus pulposus cells,using liposome analog and inhibitor of miR-328 transfection into the nucleus pulposus cells,culture after 72 hours,using flow cytometry to compare transfection group,miR-328 analogue transfection group,and miR-328 inhibitor transfection group of cell apoptosis.7)fluorescence quantitative real-time PCR method was used to detect the mRNA levels of PAK6 and proliferation-apoptosis related molecules in myeloid nucleus cells after transfectionMiR-328 analog and inhibitor transfection into the nucleus pulposus cells,culture after 72 hours,collecting cells,extraction of RNA and real time fluorescence quantitative PCR,comparison between groups of PAK6 proliferation and apoptosis related molecules,such as Bax,Bcl 2,Caspase 3,etc.The expression of mRNA level.8)protein immunoblot assay was used to detect the protein expression of PAK6 and proliferation-apoptosis-related molecules in nucleus pulposus cells after transfectionMiR-328 analog and inhibitor transfection into the nucleus pulposus cells,culture after 72 hours,collecting cells,extraction of protein in the cell,through the protein immunoblot method comparison between groups of PAK6 proliferation and apoptosis related molecules,such as Bax,Bcl 2,Caspase 3,etc.The expression of protein levels.9)dual-luciferase reporter assay verified the target relationship between mir-328 and PAK6Train 293 cells,the wild type and mutant design contains miR-328 and PAK6 3'noncoding region complementary sequence of plasmid,the analog common transfection and miR-328 to 293 cells,adopt the method of dual luciferase report experiment,verification of miR-328 and PAK6 directly targeted relationship.Results:1.Comparison of the expression of mir-328 in IDD models of normal rats and ratsThe expression of mir-328 in normal intervertebral disc rats and IDD rats was detected by rt-qpcr.Compared with the control group,the expression of intervertebral disc mir-328 was significantly increased in IDD rats(p<0.05).2.Effects of mir-328 on NP cell proliferation and apoptosisAfter transfection with mir-328 simulators,the proliferation of NP cells significantly decreased at no time point,while the proliferation of NP cells significantly increased within 24 and 48 h after transfection with mir-328 inhibitors.On the other hand,after the transfection of mir-328 analog,the apoptosis rate of NP cells increased significantly,while the transfection of mir-328 inhibitor could inhibit the apoptosis of NP cells(p<0.05).3.PAK6 is a direct target of mir-328MiR-328 analog of mutant PAK6 3 'UTR-luciferase(MUT)transfection cells the activity of no significant influence(p>0.05),and miR-328 analog can lead to wild type PAK6 3'-UTR(WT)transfection cell luciferase activity decreased significantly.These results indicate that PAK6 is a direct target of miR328.4.Mir-328 can regulate the proliferation and apoptosis of NP cells by targeting PAK6The expression of intervertebral disc PAK6 in IDD rats was significantly decreased(p<0.05).In addition,transfection of mir3-28 analogues in np-cells resulted in a significant decrease in PAK6 expression,while transfection of mir-328 inhibitors resulted in an increase in PAK6 levels.In addition,transfection miR-328 analog can lead to promote apoptosis protein caspase 3,the expression of bax increased significantly,resistance to apoptosis protein expression of BCL-2 significantly,and transfection of miR-328 inhibitor has had the opposite effect(p<0.05).Conclusion:miR-328 was up-regulated in IDD;moreover,miR-328 might regulate the proliferation and apoptosis of NP cells through targeting PAK6.The second partPanax notoginseng saponinscan alleviate intervertebral disc degeneration through regulating 1L-1?/NF-kB signaling pathwayThe research backgroundIntervertebral disc degeneration(IDD)is a common vertebral disease.In recent years,along with the accelerating pace of life and bad living habits,now the disease occurrence and development of high incidence and younger trend,there have been reports in people under the age of 30,up to 40%of the disease,and once more than 50 years old,this proportion is more will be as high as 90%.With the worsening of function of intervertebral disc,the disease will be further development for intervertebral disc herniation,spinal canal stenosis,more serious diseases such as scoliosis and great influence on the patient's physical and mental health at the same time,also can bring huge economic burden to patients.At present,the mechanism of intervertebral disc degeneration is not clear.Existing clinical treatments include conservative treatment(such as bed rest,anti-inflammatory pain relief,physical therapy)and surgical treatment.Conservative treatment can generally alleviate the pain of acute patients,but if the conservative treatment fails,surgical treatment must be carried out.Surgical treatment mainly focuses on highlighting the nucleus pulposus resection and intervertebral fusion.Surgical treatment can achieve a certain effect,but the cost is high,the risk of infection is high,and the long-term effect is uncertain.Therefore,in the field of the treatment of intervertebral disc degeneration related diseases,along with the in-depth study of related mechanism in recent years,how to build a more safe and effective new type of special treatment is a difficult problem facing the medical and scientific research workers.In recent years,the pathogenesis of intervertebral disc degeneration has been deeply discussed.Current mainstream theories suggest that advances in intervertebral disc degeneration may involve the abnormal expression of some pro-inflammatory cytokines in which interleukin-1 is widely reported.Abnormal increase expression of IL-1 beta increased to induce increase the expression of matrix metalloproteinases(MMPs),the BCL-2 antiapoptotic gene expression,which leads to the abnormal apoptosis cells and the degradation of extracellular matrix(ECM).Nf-kb is one of the most common nuclear transcription factors and plays an extremely important role in various immune responses.In the inflammatory reaction,the activation or inhibition of nf-kb and its signaling pathways will directly influence the secretion of relevant inflammatory factors and the expression of relevant signaling molecules,thus accelerating or inhibiting the process of inflammatory reaction.However,further studies are needed to elucidate the specific mechanisms involved in the development of il-1 furnish and nf-kb in intervertebral disc degeneration.Traditional Chinese medicine has long been an important means to treat intervertebral disc degeneration.In recent years,along with the deepening of the research of traditional Chinese medicine and redevelopment,the active components of traditional Chinese medicine,such as the effect of traditional Chinese medicine monomer in the treatment of intervertebral disc degeneration in there are numerous unsubstantiated reports and related mechanism research.Total saponin of panax notoginseng is the main effective component of panax notoginseng,which has been famous for its function of promoting blood circulation and removing blood stasis since ancient times.Therefore,total saponin of panax notoginseng has an excellent development prospect in the medical field.At present,according to a study by notoginseng total saponin for disease of heart head blood-vessel,pneumonia,liver disease,autoimmune diseases such as have good curative effect,and in the orthopaedic disease,notoginseng total saponin has also been widely used with fracture,bone trauma,the treatment of bone and cartilage injury.The study found that notoginseng total saponin by activating or inhibiting the cell signaling pathways,promote the formation of bone and cartilage,and animal studies,according to the results of notoginseng total saponin can effectively regulate the circulation of the blood of animals,and have the antithrombotic,inhibiting platelet aggregation and dilate blood vessels and increase blood flow.These processes can effectively repair the damage of local soft tissue,promote the healing of bone damage,increase bone density and improve the condition of osteoporosis,thus giving full play to its therapeutic effect.Cell and animal experiments are the most common methods in basic research.Among them,the establishment of animal models of various diseases plays an extremely important role in pre-clinical studies on the pathogenesis and efficacy of drugs.In intervertebral disc degeneration mechanism of through the establishment of animal model of intervertebral disc degeneration can be a good alternative and complementary,etiology and treatment for the disease to provide good research means.Over the years,researchers all over the world to explore the modeling method of the intervertebral disc degeneration in great quantities,and among them,lumbar puncture method is a simple and practical,and very reliable can fast methods of intervertebral disc degeneration model is established.At present,this method has been widely reported in the literature and has been widely recognized by researchers at home and abroad.So far,notoginseng total saponin can inhibit beta and NF-IL-1 kB related signaling pathways,in order to delay the related mechanism of intervertebral disc degeneration has not yet been reported,and as a result,in this study,we will first discuss notoginseng total saponin in the treatment of intervertebral disc degeneration in the potential role,through the establishment of animal model of intervertebral disc degeneration method,through the drug in the body and in vitro cell experiment,the method of studying the notoginseng total saponin in the treatment of degenerative lumbar potential,and its possible mechanism of action,for the clinical treatment of intervertebral disc disease patients to provide new ideas and methods for treatment.Objective:1)determine whether total saponin of panax notoginseng can slow down the degenrelated study,because of clinical specimens get difficulty,and lack of control,therefore,eration of intervertebral discs by inhibiting the expression of il-1 protein and other inflammatory factors in rat tissues and blood.2)to determine whether,at the cellular level,total saponins of panax notoginseng can inhibit the secretion of inflammatory factors and the degeneration of nucleus pulposus cells by regulating the signaling pathways related to nf-kb.Experimental method:1)establishment of animal models of intervertebral disc degeneration in ratsFemale Wistar rats were randomly divided into 5 groups of 10 rats in each group.Including sham operation group,modeling group,modeling group+panax notoginseng total saponin low,medium and high dose group.Lumbar puncture was used to establish a model of intervertebral disc degeneration in rats.After anesthesia in rats,the prone position and fixed on the operating table,using Faxitzon apparatus determine its after lumbar segment,with 20 ml syringe needle pierced its 4-5 intervertebral disc,to simulate mechanical damage caused by the intervertebral disc degeneration,and the control group only will be pulled out after needles to Pierce the skin.After two weeks of modeling,MRI was used to determine the degeneration of each rat's intervertebral disc.2)administration of total saponins of panax notoginsengAfter rat intervertebral disc degeneration model is successfully established,the control group and model group only daily lavage with saline,and notoginseng total saponin low,medium and high group respectively according to the 10 mg/kg.(d),20 mg/kg.(d),the dose of 40 mg/kg.(d)to give medicine,the whole process for 2 weeks.After the treatment,the rats were executed,their intervertebral disc tissues were removed,and the excess muscle and fat around them were removed,and the-80oc refrigerator was set aside.3)pathological examination and determination of degeneration degree in intervertebral disc tissue of ratsUsing HE staining method for each group of intervertebral disc tissue pathology examination,and the RT-qPCR and Western blot method to detect tissue type ? collagen,collagen type ?,the expression of type ? collagen,protein,polysaccharide,etc in order to determine the degree of intervertebral disc degeneration in the organization.4)expression of inflammatory factors in rat tissues and serumImmunohistochemistry and ELISA methods were used to detect the expressions of inflammatory factors such as il-1 and il-6 in different concentrations in each group and untreated group.5)isolation,primary culture and identification of nucleus pulposus cells from degenerated intervertebral disc tissue in ratsAfter rat intervertebral disc degeneration model is successfully established,the rats after anesthesia,after anesthesia in rats,the prone position and fixed on the operating table,after disinfection of skin incision,cut the lumbar spine,collecting cells,then puts its culture in a bottle,the method of using difference attached for culture and purification,and identification and training.Through cell H&E staining method for dyeing identification of nucleus pulposus cells,using immunohistochemical method in groups of type ? collagen,collagen type ?,the expression of type ? collagen,protein,polysaccharide,etc to determine the extent to which each cell intervertebral disc degeneration.9)expression verification of nf-kb signaling pathway related signal proteins in rat degenerative disc tissues and nucleus pulposus cellsCell culture supernatant solution was collected and the expressions of inflammatory factors such as il-1 and il-6 in each group were detected by ELISA.In addition,in order to define the NF-kB signal pathways are involved in this process,we will be using rt-pcr and Western blot method,respectively,inspection organization in the first part and second part testing comparison between groups of NF-kB cells and its related signaling proteins,such as the MMP-9,the expression of Bcl-2 and so on,in order to make clear the NF-whether kB related signaling pathway in notoginseng total saponin improve lumbar degeneration mechanisms play an important role.Results:1.Effects of total saponins of panax notoginseng on IDD model ratsAfter PNS treatment,the expression of type ? collagen and GAG was significantly decreased,while the expression of type ? collagen was significantly increased in mRNA and protein levels.The expressions of il-1 and il-6 in serum of each group of rats were detected by ELISA.PNS can significantly reduce the expression of il-1 and il-6 in serum of IDD rats(p<0.05),which is dose-dependent.2.Effects of total saponins of panax notoginseng on nucleus pulposus cells in vitroNucleus pulposus cells were isolated from normal rats and IDD rats,and the effect of PNS on NP cells was detected.It was found that 20 and 50mg/L PNS significantly decreased the secretion of il-1 and il-6 in the cultured supernatant solution of IDD rat nucleus pulposus cells(p<0.05).3.Effects of total saponins of panax notoginseng on the expression of NF-acetyl in medullary nucleus cells of IDD rats and IDD ratsLarge dose and high dose of PNS treatment can significantly reduce the IDD NF-kB in mice,the expression of MMP-9,at the same time in the in vitro experiments,PNS can also significantly reduce the IDD rat NP cells NF-kB,the level of MMP-9(p<0.01).PNS treatment resulted in significantly decreased expression of type I collagen and GAG in intervertebral disc tissues of IDD rats,significantly increased expression of type ?collagen,and increased expression of il-1 and il-6 in serum of IDD rats.After NP cells were cultured in vitro and treated with PNS,il-1 and il-6 in supernatant solution were also decreased.Rt-qpcr and Western blot test results showed that medium and high concentration of PNS significantly promoted the expression of bcl-2,and significantly inhibited the expression of mmp-9 and nf-kb.Conclusions:PNS can alleviate intervertebral disc degeneration through regulating IL-1?/NF-kB signaling pathway. |
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