Role Of Myeloid Differentiation Protein 2 In The Development Of Tissue Injury And Pain Behavior In Knee Osteoarthritis

Knee osteoarthritis(KOA)is a chronic degenerative disease characterized by progressive chronic pain and movement dysfunction.It is one of the most prevalent chronic diseases with an incidence as high as 21% in adults.Due to its high prevalence and rate of disability,KOA has severely affected patients’ life quality and brought heavy burden for the health system and the society.However,current treatments-cartilage nutrition,joint lubrication,local physical therapy(heating)and non-steroidal anti-inflammatory analgesic drugs – are often lack of strong clinical support or mainly aimed at symptoms with risk of develoing tolerance.They also has little effect on slowing down disease progression.At final stage when patients developed unbearable pain or severe joint deformation with movement limitation,artificial joint replacement is the ultimate solution.However this is a major procedure with multiple risks,and not to mention,very costly to patients.Therefore,it is extremely urgent to develop safe and effective therapies for KOA.Acupuncture is a valuable asset of the Chinese nation,and studies have shown that electroacupuncture can improve pain scores in patients with KOA.However,due to the inconveniences of acupuncture treatment and the international divergent opinions on the mechanism of acupuncture treatment,it has not been widely used.Therefore,it is necessary to explore the mechanism underlying acupuncture induced analgesia in KOA and maybe to find new targets for drug development.Age,diet,trauma and obesity are major risk factors for osteoarthritis.Inflammatory response is a common pathway in the above mentioned risk factors.It has been proven to play an important role in the pathogenesis of osteoarthritis.Inflammation is involved in synovial injury,cartilage degeneration,pain and hyperalgesia in KOA.Furthermore,TLR4 pathway has long been suggested as a key mediator of inflammatory reaction in KOA.Multiple small molecules that blocked TLR4 pathway can reduce chondrocyte injury.However,none of these has made it to clinical verification due to lack of specificity or limited effectiveness.Myeloid differentiation protein 2(MD2)is a pivotal accessory molecule in TLR4 pathways activation.It anchors on the outer membrane segmentation of TLR4 and provide binding sites for TLR4 which in turn facilitates TLR4 cascade signaling.Downstream NF-κB are then activated and a large quantity of inflammatory cytokine were released.However,MD2 expression and its change in the lumber spinal cord of animals with knee osteoarthritis has not been studied.No direct evidence has been published to elucidate whether MD2 participates in the hyperalgesia developed in KOA.This study is aimed to explore if MD2 is involved in the development of KOA and whether electroaupuncture interferes with MD2 to elicit its KOA theraputic effect.Furthermore,to develop a new small peptide targeting MD2 for KOA treatment.We constructed a mice model to study KOA and KOA related hyperalgesia,studied the expression change and location of MD2 in the spinal cord dorsal horn of KOA mice.Then using MD2-/-mice and lentivirus,and a small peptide specifically targeting MD2 in the nervous system(Tat-CIRP),we explored the treatment effecacy of MD2 blockade(or elimination)on KOA and KOA related hyperalgesia behavior.These research could provide new insights into the mechanism and a novel target for clinical therapy of KOA-related chronic pain and hyperalgesia.Experiment one: The therapeutic effect of electroacupuncture on MIA-induced knee osteoarthritis in miceObjective:1.To construct a mouse model of knee osteoarthritis,screen a proper dose of MIA for production of a stable model for KOA research.2.To explore the therapeutic effect of electroacupuncture on MIA-induced knee osteoarthritis in mice.Methods:1.8-week old C57BL/6 mice received intra-articular injection of 0.3mg MIA,1.0mg MIA or saline separately(MIA was dissolved in 10μl saline,n=10 in each group).Paw withdrawal threshold,weight bearing percentage,knee diameter and knee pathological OARSI scores were adopted to evaluate the hyperalgesia and damage of ipsilateral knee joint using different concentrations of MIA.2.Choosing 1.0mg MIA intra-articular injection to construct mice KOA model.Mice were randomly divided into Sham group,KOA group,KOA + EA group(n=10 in each group).Paw withdrawal threshold,weight bearing percentage,knee diameter and knee pathological OARSI scores were adopted to evaluate the hyperalgesia and damage of ipsilateral knee joint at 7d and 14 d after intra-articular knee MIA injection.Results:1.The pain behavior,arthrocele and pathological damage of 1.0mg MIA group are more significant and more stable than those of 0.3mg MIA group.No changes in knee pathology or pain behavior was detected in saline injection group.2.Compared to the KOA group,the pain behaviors(paw withdrawal threshold and weight bearing percentage)of the MIA+EA group were significantly improved.Conclusion:1.1.0mg MIA intra-articular injection was selected to construct mice KOA model for further research.2.Electroacupuncture can improve the pain behavior of MIA-induced knee osteoarthritis in miceExperiment two: MD2 in the spinal cord dorsal horn is involved in the therapeutic effect of electroacupuncture in knee osteoarthritisObjective:1.To elucidate the expression and cell localization of MD2 in the spinal cord dorsal horn of mice at different stages of knee osteoarthritis.2.To explore whether MD2 is involved in the therapeutic effect of electroacupuncture on knee osteoarthritis.Methods:1.Western-blot as well as immunofluorescence staining were used to analyze the expression of MD2 in mice spinal dorsal horn at 6h,12 h,1d,3d,7d and 14 d after intra-articular knee MIA injection.Double immunofluorescence staining was adopted to investigate the cell localization of MD2 at different time points and in different types of cells including neuron,microglia and astrocyte.2.Western Blot was used to detect the expression changes of MD2 of KOA mice on day 7 after electroacupuncture treatment.Using MD2 over-expression lentivirus to raise the expression of MD2 in the spinal dorsal horn.Paw withdrawal threshold,weight bearing percentage,knee diameter and knee pathological OARSI scores were adopted to evaluate the hyperalgesia and damage of ipsilateral knee joint at 7d and 14 d after intra-articular knee MIA injection.Results:1.Both the Western-blot and immunofluorescence staining results showed that the expression of MD2 was up-regulated in both ipsilateral and contralateral in the lumbar spinal cord dorsal horn at 3d,reached peak at 7d and reduced at 14 d after MIA injection.Besides,double immunofluorescent staining showed that at 7 days after MIA injection,MD2 was mainly produced in neurons,but not microglia or astrocytes in the spinal cord dorsal horn.2.Western-blot results showed that the up-regulated peak values of MD2 on both side of spinal dorsal horn(7d after MIA injection)were significantly reduced after the treatment with electroacupuncture.Meanwhile,compared with the KOA+EA+LV-Con group,the pain degree of the KOA+EA+LV-MD2 group was significantly aggravated.Conclusion:1.The expressions of MD2 in both sides of the spinal cord dorsal horn in the lumbar enlargement are significantly increased in early stage of knee osteoarthritis,peaks at 7 days and reduced by 14 days after MIA injection.The increased MD2 expression was mainly located in neurons.2.Electroacupuncture can significantly inhibited the MD2 upregulation on day 7 after MIA injection,and the re-overexpression of MD2 in spinal cord dorsal horn with lentivirus reverses the analgesic effect of electroacupuncture.Experiment three: Effects of genetic deletion or specific blockade MD2 on knee osteoarthritis and related pain behaviorObjective:To explore whether MD2 knock-out or specific functional inhibition could reduce pathological changes and pain behavior in mice with knee osteoarthritis.Methods:1.Eight-week old wild type(WT)or MD2 knockout mice(n=10 each)were used.Paw withdrawal threshold,weight borne,knee diameter were used to analyze the hyperalgesia and knee inflammation before and at 1,3,7 and 14 days after MIA injection.Ipsilateral knee pathological changes were assessed after the last behavioral test at 15 days after MIA injection.2.The injection of virus may specificity down-regulate the expression of MD2 in the spinal cord dorsal horn.Paw withdrawal threshold,weight bearing percentage,knee diameter and knee pathological OARSI scores were adopted to evaluate the pain and damage of ipsilateral knee joint between MD2-si RNA and Con-si RNA.3.30 mg/kg of MD2 specific inhibitor-peptide,Tat-CIRP,was injected intravenously once per day for 7 days after MIA injection to explore treatment effect of MD2 blockade on KOA and related hyperalgesia.Outcome measurements are similar to part 1.4.Direct intrathecal delivery of 3 mg/kg Tat-CIRP once per day at 1,3 and 5 days after MIA injection was assessed for effects on KOA and related hyperalgesia behavior.To rule out the effect of Tat-CIRP transferring from cerebral spinal fluid into the peripheral blood stream,another two groups in which 3 mg/kg of Tat-CIRP was injected intravenously at same time intervals were also assessed.Outcome measurements are similar to part 1.Results:1.The pain related behavior(PWT and weight borne),arthrocele and knee joint pathological damage were all improved in MD2 KO mice compare to heterozygous littermates group and wild type group.No difference between the latter two groups were observed.Whole body knockout of MD2 can reduce KOA pathological change and reduce KOA related pain behavior including paw hyperalgesia.2.Compared with Con-si RNA group,the pain performance of mice in the MD2-si RNA group was partially improved,the degree of joint swelling was not significantly changed,and there was no significant difference between the affected downregulation group(ips)of and the bilateral downregulation group(ips+con).3.The pain related behavior(PWMT and weight borne),arthrocele and knee joint pathological damage were all ameliorate in mice administered Tat-CIRP intravenously 30 mg/kg per day for 7 days after MIA injection.These results indicate that intravenously delivery of specific MD2 blockade peptide could reduce both KOA knee damage and pain related behavior in KOA mice.4.Intrathecal delivery of low dose Tat-CIRP(3 mg/kg)significantly reduced PWT in the KOA mice(P=0.0436),affected but not significantly reduced weight bearing deficit(P=0.065),while arthrocele and knee pathology did not improve by intrathecal Tat-CIRP administration.Intravenous low-dose Tat-CIRP did not improve arthrocele,knee pathology,neither did it provide reduction in hyperalgesia compared to saline control.These results indicate that direct inhibition of spinal MD2 function could provide analgesia(reduced hyperalgesia)without affecting knee pathological changes.Spinal cord MD2 is a key mediator in hyperalgesia development in KOA model.Conclusion:Genetic deletion and specific blockade of MD2 function throughout the body could provide KOA joint protection as well as pain relieve.However,specific down-regulation or intrathecal blockage of MD2 could only reduce hyperalgesia(PWT),but did not affect knee pathology.These results indicate that MD2 in the spinal cord is involved in the formation of hyperalgesia but not arthrosis in the model of knee osteoarthritis.And MD2 can be a new target for KOA treatment.Our MD2 targeting peptide provides a prominent new drug for KOA therapy.