Effects Of Gu Ling Gao And Its Split Prescription Preparation To Cartilage Cell Apoptosis And Extracellular Matrix Degradation On Osteoarthritis

Gu Ling Gao(GLG) is compound preparation composed of RhizomaDrynariae，Radix Clematidis，Angelica，Chuanxiong Rhizome，MorindaRoot，Radix Rehmanniae Preparata，Chinese Wolfberry etc.and multiflavor Traditional Chinese Medicine prescription compatibility，Is following the traditional medical treatment of arthralgiasyndrome that modern medicine called osteoarthritis (OA) ofreinforcing the liver and kidney, strengthening tendons and bones，promoting blood circulation and removing blood stasis，expellingwind and removing dampness and many other rule on the basis oftheoretical preparation，Put forward a“Warming Yang and Invigora-ting Qi，nourishing yin and enrich the blood”rule of treatmentby nourishing liver and kidney function recovery of decadentapraxia bones，tonifying spleen recovered bones and the source ofQi and blood biochemistry，restore imbalances viscera function“visceral disease”through conditioning heart lung make main andcollateral channels gas comfortable machine and treatment of“collateral disease”that pathogenesis is main and collateralchannels and Qi and blood tangible stasis by“promoting bloodcirculation to remove meridian obstruction”united Theory,At the same time,comparative study of the role on OA according to Ling Gaoand Gu Gao preparation In accordance with the theory of“collateraldisease”and“visceral disease”proceed separation of prescriptioncompatibility, integration of the theory treatment of OA, in orderto establish a unified treatment of thinking for clinical treatmentof OA disease.OA is a kind of articular cartilage degeneration and secondarybone hyperplasia is characteristic of chronic joint disease.Manystudies show that the disease and the body by the naturalenvironment changes such as cold and wet stimulation,The bone andjoint system load and local biological change, the imbalance ofimmune function,obesity and endocrine metabolic disorders, relatedto drug and food improper factors,The lesions in the weight-bearinglarger knee, hip, spine and distal interphalangeal joints, thedisease also known as bone and joint disease, degenerativearthritis,hypertrophic arthritis etc.The earliest and mostPathological changes of OA beginning of the articular cartilage,gradually affecting the entire joint structure，including thesubchondral bone，synovial tendon，ligament，joint capsule andsurroundingmuscle tissue, eventually cause joint deformity andloss of function for all loss of articular cartilage.From the resultof OA pathological change is a slow development and gradual process,therefore making the experimental animal model must followthispathological changes, now making animal model of OA inducedmethods such as local intra-articular injection of drugs andimplanted foreign bodies, local joint damage of Hulth operationmethod and by natural selection, mutationgenetic breeding ofspontaneous OA animal model can not well reflect this process, and the use of joint cold stimulation method combined with thelocaljoint plaster immobilization can very good expression andreduction of the pathological process, provide the animal model iscloser to human clinical history for the experimental research ofOA, therefore this experiment adopts"the cold stimulation combinedwith joint fixed-cold consolidation" making experimental animalmodel.The studies showed that OA lesions characterized by articularcartilage degeneration, and the apoptosis of chondrocyte andextracellular matrix-degradation is main based and cause onmolecular biology of cartilage degeneration, oxidative injury,inflammation, immune dysfunction inthis disease always, plays animportant role, at present for the treatment of the disease,clinical is often limited to simple anti-inflammatory,orantioxidant, or enhance immune function, or improve the cycle ofone or several aspects collaborative treatment, not the wholetreatment up to the level of theory.Therefore, the purpose of thisstudy is to study on the molecular basis of the chondrocyteapoptosis and cell extracellular matrix degradation path onprescription preparation bone plaster and its disassembledprescriptions preparation bone paste, plaster and Western medicinecelecoxib to OA animal model induced for cold consolidation ofoxidative damage,inflammation, immune function changes of, use ofmodern molecular biology technique, to explore the boneplaster andits demolition preparations for the OA animal model of articularcartilage degeneration mechanism, and to explore the protectiveeffect and mechanism of traditional Chinese medicine on jointdamage in animal models of OA through comparative study onpreparation of TCM and Western medicine, which provides theoretical and experimental basis for Gu Ling Gao treatment of OA, To lay thetheoretical foundation about thought of Combined treatment of OArise Overall treatment and overall adjustment to“collateral andvisceral disease”.Experiment One Rat osteoarthritis animal model preparationby cold consolidation methodObjective: to establish an experimental animal model isconsistent with the clinical etiology of human osteoarthritisdisease.Methods: experimental rats were divided into model groupand normal control group, model group rats were given daily (6±0.5)℃cold water stimulationcombined with joint plasterfixation in each4h,a total of6weeks; control grouprats were giventhe same spatial variation, duration,no the molding operation.Bodyweight and joint diameter measurement in rat after6weeks, therat serum,knee joint cartilage and synovium, articular cartilagepathological changes were observed under the microscope, X todetect changes of joint imaging,ELISA detection of serum TNF-a,IL-1β, HA, IgM content, detection of articular cartilage andsynovial cell apoptosis by TUNEL.Results: the cold and solid modelsof OA were made in X imaging, joint swelling, pathological injuryand apoptosis of cartilage and biochemical changes are in line withthe characteristics of OA, indicating that the model building wassuccessful.Conclusion:the application of cold stimulationcombined with joint fixation for6weeks can make bone arthritisexperimental animal model well, detection index and signs of modelanimal and the laboratory line with human osteoarthritisclinicaldiagnosis. Experiment Two Effect of Gu Ling Gao and Remove the PartyPreparations on osteoarthritis oxidation damageObjective:To observe the effect of Gu Ling Gao(GLG),GuGao(GUG),Ling Gao(LG)、celecoxib(CLXB) against OA oxidative damagedifferences, to explore the mechanisms of different compatibilityagent on OA.Methods: after the model of SD rats were randomlydivided into GLG group, GUG group, LG group, CLXB group, M groupand NG group were divided into6groups, GLG, GUG and LG groups weregiven corresponding preparations and in accordance with the10.41ml·kg-1·D-1(equivalent to the dosage of33.31g·kg-1)byintragastric administration, CLXB group was given CLXB20.83mg·kg-1·D-1Ig, M and NG were given normal saline, daily by gavage1times, a total of8weeks. Take serum,articular cartilage tissueof rat, testing the content of SOD, MDA, LPO in serum by ELISA,testing the positive expression of HIF-1a, HIF-2a, VEGF withimmunofluorescence method, to detect the expression of HIF-1a,HIF-2a, VEGFmRNA with FQ-RT-PCR.Results:M group compared with NGgroup and GLG group could significantly increase the content ofserum MDA, LPO (p<0.01),increased the content of SOD(p<0.01); GLGcompared with GUG and LG can increase the contents of SOD(p<0.01),decrease the content of MDA,LPO(p<0.01);M group comparedwith NG group can increased the positive rate of HIF-2a，VEGF，HIF-1a(p<0.01)significantly in Cartilage,GLG compared with M，GUG，CLXB，LG group can low positive rate of HIF-1a，HIF-2a，VEGF(p<0.01)；M group compared with NG group could significantly increase theexpression of HIF-1a，HIF-2a，VEGFmRNA on cartilage(p<0.01)，GLGcompared with M，GUG，CLXB，LG reduced expression of HIF-1a，HIF-2a，VEGFmRNA(p<0.01).Conclusion:GLG can through increase the contentof serum SOD, reduce the content of LPO, MDA, to correct OA oxygen metabolism imbalance cause oxidative damage to the cartilage tissue,inhibit expression of HIF-1a，HIF-2amRNA，to inhibit abnormalhypoxia environment HIF-1a to HIF-2a conversion, decreased theexpression of downstream target gene VEGFmRNA in cartilage tissue,reduce the formation of joint osteophytes, against of cartilageinjury on OA, and the effect was better than that of CLXB, LG,GUG.Experiment Three Effect of Gu Ling Gao and Remove the PartyPreparations on osteoarthritis inflammation injuryObjective:To study the GLG and Remove the PartyPreparations(RPP)on effect of related inflammatory cytokines onOA，to explore its protective effect of inflammatory injury onOA.Methods:cold consolidation molding and after administration，take rats serum， synovial and cartilage tissue， ADAMTS-4,5expression was detected by Western blot，the content of IL-1β，IFN-γ，TNF-a，PGE2，COX-2on synovial membrane and serum with IL-18，IL-17on serum was tested by ELISA，positive expression of IL-18，IL-17on cartilage was tested by immunofluorescenc.Result:(1)Mgroup compared with NG group and GLG group could increase contentof IL-1β,TNF-a,PGE2, COX-2,IFN-γ(p<0.01)on serum and synovialsignificantly；GLG compared with GUG could decrease the content ofIL-1β，TNF-a，COX-2，IFN-γ(p<0.01)on serum and IL-1β，COX-2，PGE2(p<0.01)on synovial；GLG compared with LG can reduce thecontent of TNF-a，PGE2，COX-2，IFN-γ(p<0.01)on serum and synovial；GLG compared with CLXB could decrease the content of IL-1β， TNF-a，PGE2，IFN-γ(p<0.01)on serum and IL-1β，PGE2on synovial(p<0.01).WB results showed that the ADAMTs-4，5had expression in each group，among which the M group was most significant；GLG group；ADAMTs-45protein minimum expression.(2)M group compared with NG group could increase positive expression of IL-17，IL-18in cartilagecells and content of IL-17，IL-18in serum significantly(P<0.01)，GLG compared with M，GUG，LG and CLXB group can significantly reducethe positive expression and content of IL-17，IL-18(P<0.01) oncartilage cells and serum.Conclusion:(1) GLG may regulate bodyimmunity inflammatory cytokines IFN-γ While reduced relatedinflamma-tion cytokines content of IL-1β，TNF-a in serum andsynovial on OA.Then stop the inflammatory mediators PGE2，COX-2influence on ADAMTs-4，5protein anabolism in local articularcartilage，reduce OA cartilage injury，its effect is better thanthe disassembled prescription preparation andcelecoxib.(2)GLG maythrough reduce content of IL-17，IL-18in serum and positiveexpression on cartilage cells，combat Inflammatory damage in OA，its effect is better than the disassembled prescription preparationGUG，LG and CLXB.Experiment Four Effect of Gu Ling Gao and Remove the PartyPreparations on immune function of osteoarthritisObjective：To investigate GLG and RPP on the mechanism of immunefunction on OA by cold consolidation induced.Methods：cold solidmodeling and administration, take rat plasma, knee joint synoviumand cartilage tissue, test positive cells rate of CD3+，CD4+，CD8+of plasma and synovial by immunofluorescence；test percent rate ofCD3+，CD4+，CD8+and CD80，CD86，Col-Ⅱ on cartilage by flowcytometry.Results：M compared with NG group decreased percentageof CD3+，CD4+in plasma and CD3+，CD4+，CD8+T lymphocyte oncartilage(P<0.01)，Increased percentage of CD3+，CD4+，CD8+，CD4+/CD8+T lymphocyte in synovium and positive percentage of CD80，CD86，COL-II in cartilage(P<0.01)；GLG compared with M group can rise percentage of CD3+，CD4+T lymphocyte in plasma and cartilagein rats OA, increased the ratio of CD4+/CD8+(P<0.01),decreasedpositive percentage of CD80，CD86，COL-Ⅱ in cartilage(P<0.01),decreased ratio of CD3+, CD4+, CD4+/CD8+in synovial membrane(P<0.01);GLG compared with GUG， LG， CLXB group can increasepercentage of CD3+，CD4+T lymphocyte in plasma and cartilage,increased the ratio of CD4+/CD8+(P<0.01).Conclusion: GLG maythrough improve the overall and local joint immune function in arat model of OA，inhibition the activation of chondrocytes APCfunction and immune responses against self antigens，reducinginflammatory immune response in synovial， protection of OAcartilage and synovial membrane，the effect is better than thedisassembled prescription preparation and celecoxib.Experiment Five Effect of Gu Ling Gao and Remove the PartyPreparations on cartilage cell viability of OAObjective: To investigate the mechanism of GLG and RPP with CLXBon OA cartilage cell proliferation activity of OA.Methods: coldsolid modeling and administration，treated cartilage cells from OArats were cultured in vitro with each rats serum；mirror box countingcell density，identification of cartilage cells by immunofluoresc-ence detection the expression of type II collagen，light absorptionvalue of cartilage cells were detected with CCK8method，collectspleen and thymus of rats in each group to calculate theindex.Results：the density of chondrocyte was handled throughmedicated serum of GLG etc was higher than that in M group (P<0.01);Mcompared with NG group can reduce the light absorption value ofchondrocytes was cultured in vitro significantly(P<0.01),cellproliferation activity was decreased; GLG compared with M，GUG， LG，CLXB group can increase the OD of cartilage cells(P<0.01)，andenhanced cell proliferation;M compared with NG group can reducethe SI， TI of experimental animal significantly(P<0.01);GLGcompared with M，GUG，LG and CLXB group can increase the SI,TI ofexperimental animal(P<0.01).Conclusion:GLG promote cell different-iation may by enhanced spleen and thymus functions on OA model ratsbody，promote local cartilage cells proliferation activity，topromote the regeneration of cartilage tissue after joint injury，block articular cartilage degeneration of OA, protect articularcartilage，its effect is better than the disassembled prescription-preparation GUG，LG and CLXB.Experiment Six The influence of GLG and RPP on extracellularmatrix degradation and chondrocyte apoptosis on OAObjective:To investigate the mechanism of extracellular matrixdegradation and Chondrocyte apoptosis on OA through GLG andRPP.Methods: After cold solid modeling and administration,(1) Takethe cartilage tissue of rat knee joints，by hematoxylin and eosin(HE),safranin fast green(SA/FG),safranin alcian blue (SA/AB) forhistopathological observation respectively，Proceed grading systemto articular cartilage pathology according to the improved Mankinscore，the expression of CHOP/GADD153protein was detected byWestern blot method,detecting the expression of Bcl-2，Bax，MMP-3，TIMP-1mRNA on cartilage cells by fluorescence quantitative PCR.(2)Take cartilage tissue of knee joint in rats，the content of Wnt-2，β-catenin in cartilage tissue was using immunohistochemicaldetection，the expression of LRP-5using Western blot methoddetection,the expression of Caspase-3，BMP-2，MMP-13，Caspase-9mRNAon cartilage cells was detected by RT-PCR.(3)Take knee cartilage tissue and serum of rat,The positive expression of type II collagenand contect of NO，IL-10in serum was detected by immunofluorescenceand ELISA respectively method.Result:(1)compared with the NG group,M group could reduce the expression of TIMP-1，Bcl-2mRNA(P<0.01)and increase the expression of Bax， MMP-3mRNA(P<0.01)significantly.Compared with the M group，GLG can downregulate theERS associated CHOP/GADD153protein generation(P<0.01)，promotethe anti-apoptosis suppressing gene expression of Bcl-2mRNA anddecreasing the expression of apoptosis gene BaxmRNA (P<0.01)，andthe up regulation of Bcl-2/bax，inhibition abnormal apoptosis ofcartilage cells;at the same time，GLG can decreased expression ofMMP-3mRNA than M significantly，increased expression of TIMP-1mRNA(P<0.01)， reduced MMP-3mRNA/TIMP-1mRNA.(2) GLG can reduce OAcartilage tissue superficial to calcified each layers ofpathological injury; GLG and RPP can down-regulation the positiveexpression of Wnt2，β-catenin significantly，GLG compared withGUG，LG positive rate of β-catenin were decreased significantly(P<0.01).Western Blotting showed that the expression of LRP-5protein was decreased in GLG group than other groups significantly；FQ-RT-PCR showed that the expression of BMP-2mRNA in GLG group wasincreased than GUG group significantly(p<0.01)，and compared withLG was decreased significantly(p<0.01)， had no significantdifferences compared with NG group(P>0.05);GLG and RPP comparedwith M group the expression of BMP-2， MMP-13， Caspase-3，Caspase-9mRNA were decreased(p<0.01)，GLG compared with LG，GUG，CLXB the expression of MMP-13，Caspase-3，Caspase-9mRNA wasdecreased significantly(p<0.01).(3)M compared with NG group couldsignificantly reduce the positive rate of type II collagen and the content of IL-10in serum (P<0.01)；GLG compared with M， GUG，LG and CLXB group could increase the positive rate of type IIcollagen and the content of IL-10in serum (P<0.01) Signific-antly；M compared with NG group can increase the content of serumNO (P<0.01)significantly；GLG compared with M，GUG，LG and CLXB groupcould decrease the content of NO in serum significantly(P<0.01).Conclusion:(1)The mechanism of GLG against Chondrocyte apoptosisand Extracellular matrix degradation on OA:①GLG through reducingabnormal generation of ERS pathway related protein GADD153, promotethe expression of antiapoptosis gene Bcl-2mRNA and reduceexpression of apoptosis gene BaxmRNA,raise the ratio of Bcl-2/baxand inhibit abnormal apoptosis of cartilage cells；then GLG byreduce expression of MMP-3mRNA， increase expression ofTIMP-1mRNA，decrease ratio of MMP-3mRNA/TIMP-1mRNA then prevent theabnormal degradation of extracellular matrix.②GLG may be throughbalance adjustment the expression of BMP-2mRNA on OA,downregulation the content of LRP-5transmembrane protein was dependenton Wnt/β-catenin Signal pathway，Stop Wnt/β-catenin signalingpathway excessive suppression and abnormal activation，then reduceexpression of MMP-13，Caspase-9，Caspase-3mRNA to lessen thecartilage degradation of ECM and abnormal apoptosis of thecartilage cell.③GLG may be reduce Oxidative damage of serum NO andenhanced the effect of anti-inflammatory with IL-10on the modelrats，Through anti-inflammatory，antioxidant pathway Inhibitiondegradation of collagen of OA damaged cartilage extracellularmatrix.(2)The Comparison about protective effect of GLG and RPPwith CLXB to cartilage injury on OA：GLG through reducing theapoptosis of OA chondrocytes and extracellular matrix degradation way to prevent the degeneration of articular cartilage，the effectprotect articular cartilage was superior to GUG，LG and CLXB.