The Effect And Mechanism Of GPR4 Regulating Nucleus Pulposus Degeneration Via Promoting CAMP Accumulation Under The Acidic Environment Of Degenerated Intervertebral Disc

Degenerative disc disease(DDD)is a common disease in clinical practice,which is the main cause of neck,shoulder,low back and leg pain.The data shows,the prevalence of DDD in China has increased year by year,and it has become an important disease that seriously affect the health status and health-related quality of life of Chinese people.At present,the treatment methods for DDD include conservative drug treatment and surgery.Unfortunately,these treatments fail to address the pathophysiological process of intervertebral disc degeneration(IDD),resulting an unsatisfactory long-term efficacy.In recent years,biological treatment,including cell transplantation and gene therapy,has become the research focus of DDD treatment.The objective of these methods is to prevent or reverse the process of IDD in the early stage.It has now been shown that IDD originates in the nucleus pulposus.The most important pathological feature of IDD is the reduction in the number of nucleus pulposus cells and the imbalance of extracellular matrix anabolism and catabolism,which is characterized by nucleus pulposus apoptosis,decrease synthesis of extracellular matrix(mainly collagen ? and aggrecan)and the increased degradation of extracellular matrix.Inflammation has been demonstrated to play a key role in IDD.Based on this pathological feature,researchers attempt to repair the degenerated nucleus pulposus using biological methods,such as cell transplantation and growth factor injection.However,it is disappointing that the results of these studies indicate that current biological methods still fail to prevent or reverse the process of IDD.The repair effect is far from the predetermined goal,and there is still a long way to go before its clinical application.Intervertebral disc is the largest avascular tissue in human body,and it is a specific acidic environment in disc in vivo.Recent research demonstrated the acidic environment in degenerative intervertebral disc is also a key factor resulting nucleus pulposus degeneration.Due to the hypoxic status in intervertebral disc,its energy is generated mainly through anaerobic glycolysis.The metabolite of anaerobic glycolysis lactate accumulates in the disc,which gives rise to a relative lower pH in intervertebral disc(pH 5.7 to 7.2),when compared to the internal environment of human body(pH 7.35 to 7.45).The pH of disc will further decrease when IDD occur,as the pH can be 5.5 in seriously degenerated disc.A number of studies have showed that the acidic environment in degenerative intervertebral disc inhibits the synthesis of extracellular matrix.What is more important is acidic pH promotes the degradation of extracellular matrix.As a result,in opinions of some scholars,degenerative acidic environment is the main "bottleneck" for the biological treatment of IDD,which restricting the repair effect of cell transplantation.Therefore,it is urgent to find out the key regulator of nucleus pulposus degeneration in the acidic environment of degenerative intervertebral disc,and clarify the related mechanism.Finally,we can effectively inhibit this factor to prompt the efficiency of the biological treatment for intervertebral disc degeneration.Ovarian cancer G protein-coupled receptor 1(OGR1)and its related subfamily are new found G-protein-coupled receptors,and has been proposed as are sensitive to H+.There are four members in this subfamily,including OGR1,G protein-coupled receptor 4(GPR4),G2A and T cell death-coupled gene 8.In the acidic environment,the receptors such can sense extracellular H+ and mediate multiple signaling pathways in cells,which participate in immune and inflammatory responses.Among these four members,OGR1 and GPR4 widely distribute in the human body,they are found in airway epithelial cells,ovarian cells,vascular endothelial cells and kidney cells.It has been determined that GPR4 is the most sensitive to H+member in OGR1 subfamily.In an acidic environment,GPR4 can up-regulate interleukin(IL)-1?,IL-6 and tumor necrosis factor alpha(TNF-a)by promoting intracellular accumulation of cyclic adenosine monophosphate(cAMP)oravascular calcium ion mobilization.Finally,the inflammatory mediator,such as TNF-a,initiates a local inflammatory response in the cells.Numbers of studies indicated that,cAMP acts as a second messenger in many cell types,which can activate the downstream mitogenic activated protein kinase(MAPK)signaling pathway.Although the exact pathogenesis of nucleus pulposus degeneration has not been fully elucidated,there has been found a large number of inflammatory mediators in degenerative nucleus pulposus tissue,and inflammation has been considered to be one of the main factors leading to nucleus pulposus degeneration.Funded by the National Natural Science Foundation of China(81501908),we explored the role of GPR4 in nucleus pulposus degeneration,and found that EIDIP,a specific inhibitor of GPR4,can inhibit the intracellular accumulation of cAMP in nucleus pulposus cells and also the expression of inflammatory mediator,IL-1? and TNF-?.Based on the fact that acidic environment(high H+concentration)of degenerative intervertebral disc and inflammation are two key factors for nucleus pulposus degeneration,and GPR4 is a specific H+sensitive receptors that mediate inflammation,we propose the scientific question:under the acidic environment of degenerative intervertebral disc,GPR4 activates MAPK signaling pathway by mediating intracellular cAMP accumulation,which initiates inflammation,inhibits extracellular matrix synthesis and promotes extracellular matrix degradation in nucleus pulposus cells.Until now,there is no relevant study reported.In order to solve the above scientific problem,we will complete the current study,which includes three parts:(1)The effect of GPR4 in regulating nucleus pulposus degeneration under the acidic environment of degenerative intervertebral disc in vitro;(2)The specific molecular mechanism of GPR4 in regulating nucleus pulposus degeneration through mediating intracellular cAMP accumulation;(3)The specific role of GPR4 in regulating nucleus pulposus degeneration in vivo.Part 1 The effect of GPR4 in regulating nucleus pulposus degeneration under the acidic environment of degenerative intervertebral disc in vitroObjective:To elucidate the specific role of GPR4 in the regulation of extracellular matrix synthesis,the expression of inflammatory mediators and extracellular matrix degrading enzymes in nucleus pulposus cells under the acidic environment of degenerative intervertebral disc in vitro.Methods:Nucleus pulposus cells were isolated from SD rat by enzyme digestion.Culture media with pH values of 7.2,6.8 and 6.4 were prepared to simulate the acidic environment of normal,mild degeneration and severely degenerated intervertebral discs.The expression of GPR4 was detected by Real-time fluorescent polymerase chain reaction(Real-time PCR)and Western blot after cultured in the three acidic conditions for 7 days.GPR4 expression was overexpressed and silenced by gene transfection and RNA interference,and the cells were cultured in the three acidic conditions for 7 days.The expression levels of aggrecan,collagen ?,IL-1?,TNF-?,inducible nitric oxide synthase(iNOS),matrix metalloproteinase-3,13(MMP-3,13)and a disintegrin and metalloproteinase with thrombospondin motifs-5(ADAMTS-5)were detected by Real-time PCR.The expression levels of aggrecan and collagen ?were evaluated by immunofluorescence staining and immunohistochemistry staining.The secretion level of glycosaminoglycans(GAG)was detected by alcian blue staining.Results:The results of Real-time PCR and Western blot demonstrated a low expression level of GPR4 under the acidic environment of normal intervertebral disc(pH 7.2),but an increased expression level in the acidic environments of degenerative intervertebral disc(pH 6.8 and 6.4).The result of Real-time PCR also indicated that GPR4 overexpression resulted in the inhibition effect on the expression of aggrecan and collagen ? under the acidic environment of degenerative intervertebral disc,and promoting effect on the expression of IL-1?,TNF-?,iNOS,MMP-3,13 and ADAMTS-5.On the opposite,GPR4 silence increased the expression levels of aggrecan and collagen ?,and inhibited the expression of IL-1?,TNF-?,iNOS,MMP-3,13 and ADAMTS-5.Immunofluorescence and immunohistochemical staining further confirmed that GPR4 overexpression would inhibit the synthesis of aggrecan and collagen ? in the acidic environment of degenerative disc.On the contrary,GPR4 silence would enhance the synthesis of aggrecan and collagen ?.Alcian blue staining showed that GPR4 overexpression resulted in a decreased GAG secretion level in the acidic environment of degenerative intervertebral disc,while GPR4 silence prompted GAG secretion.Conclusions:This result showed that the expression of GPR4 was significantly increased under the acidic environment of degenerative intervertebral disc.GPR4 inhibited the synthesis of extracellular matrix in nucleus pulposus cells and promoted the expression of inflammatory mediators and extracellular matrix degrading enzymes.Part 2 The specific molecular mechanism of GPR4 in regulating nucleus pulposus degeneration through mediating intracellular cAMP accumulationObjective:To elucidate the specific molecular mechanism of GPR4 mediating cAMP accumulation and regulating nucleus pulposus under the acidic environment of degenerative intervertebral disc.Methods:Culture media with pH values of 7.2,6.8 and 6.4 were prepared to simulate the acidic environment of normal,mild degeneration and severely degenerated intervertebral discs.Nucleus pulposus cells were cultured in the three acidic conditions,intracellular cAMP accumulation was determined by enzyme immunoassay(EIA).Culture medium of pH 6.4 was used to simulate the acidic microenvironment of severely degenerated intervertebral discs,and the nucleus pulposus cells were cultured in the medium for 24 h.Phosphorylation levels of c-Jun N-terminal kinase(JNK)and p38 MAPK were detected by Western blot.Nucleus pulposus cells with GPR4 silence and overexpression were cultured under pH 6.4 for 24 h,and the phosphorylation levels of JNK and p38 MAPK were detected.Nucleus pulposus cells were treated with cAMP inhibitor SQ 22536 and protein kinase A(PKA)inhibitor H-89,and the phosphorylation levels of p38 MAPK and JNK were measured by western blot.Nucleus pulposus cells with GPR4 overexpression were treated with JNK specific inhibitor SP600125 and p38 MAPK specific inhibitor SB203580,and the expression levels of IL-1?,TNF-?,iNOS,MMP-3,13 and ADAMTS-5 were detected by enzyme-linked immunosorbent assay(ELISA).Results:Under the acidic environment of degenerated intervertebral disc,the intracellular cAMP accumulation of nucleus pulposus cells increased with pH value decreased,meanwhile,GPR4 overexpression would lead to the increased intracellular cAMP accumulation.Phosphorylation of JNK and p38 MAPK were increased under the acidic environment of severely degenerated intervertebral discs.GPR4 overexpression further promoted the phosphorylation of JNK and p38 MAPK,while GPR4 silence inhibited the phosphorylation of JNK and p38 MAPK.In addition,inhibition of cAMP and PKA activity also inhibited phosphorylation levels of JNK and p38 MAPK.The result of ELISA demonstrated that inhibitors of JNK and p38 MAPK resulted a decreased expression level of IL-1? TNF-?,iNOS,MMP-3,13 and ADAMTS-5 in GPR4 overexpressing nucleus pulposus cells.Conclusions:Under the acidic environment of degenerative intervertebral disc,GPR4 mediated intracellular accumulation of cAMP in nucleus pulposus cells,which activated JNK and p38 MAPK signaling pathways,and then regulated nucleus pulposus degeneration.Part 3 The specific role of GPR4 in regulating nucleus pulposus degeneration in vivoObjective:To validate the specific role of GPR4 in regulating nucleus pulposus degeneration in vivo using a rat intervertebral disc degeneration model.Methods:Seventy-two male SD rats were randomly divided into four groups:normal control group(no manipulation),degenerative control group(percutaneous needle puncture),PBS injection group(percutaneous needle puncture,PBS injection)and GPR4 treatment group(percutaneous needle puncture,GPR4 shRNA injection).Each group was subdivided into 4,8 and 16 weeks 3 follow-up time points,with 6 rats at each time point.A rat model of intervertebral disc degeneration was establishment by percutaneous needle puncture.Disc height was measured by molybdenum target X-ray and the disc height index(DHI)was calculated.The water content of nucleus pulposus was evaluated by magnetic resonance imaging(MRI)with T2-weighted imaging.The effect of GPR4 silence on the delay effect of nucleus pulposus was evaluated by histological analysis by HE staining and safranine-O staining.Results:The expression level of GPR4 was significantly decreased at day 3 after GPR4 shRNA injection.The molybdenum target X-ray and MRI results at different time points suggested that the decrease of intervertebral disc height and water content of nucleus pulposus were most obvious in the PBS injection group,followed by the degenerative control group,and the decrease in the GPR4 treatment group was the lightest.Histological HE and Safranin O staining results further verified that the GPR4 treatment group had the most minor nucleus pulposus degeneration.Conclusions:GPR4 shRNA in vivo injection can effectively delay the process of nucleus pulposus degeneration.GPR4 is a key molecules regulating nucleus pulposus degeneration.