WISP-2 Induces Apoptosis Of Chondrocytes Through PPAR ? In DDH Rats

Introduction:Developmental dysplasia of the hip(DDH)is a common developmental disorder in pediatric orthopaedics.Improper treatment can lead to osteoarthritis(OA)in adolescents and young people.In the early stage of DDH,chondrocyte apoptosis plays an important role in promoting acetabular cartilage degeneration and accelerating the development of DDH.Therefore,the prevention of chondrocyte apoptosis may be an important strategy to improve the prognosis of DDH.Wnt1 induced signaling pathway protein 2(WISP-2)is a member of the connective tissue growth factor / cysteine rich 61 / nephroblastoma high expression(Nov)(CCN)family.Masatoshi et al.Reported that the expression level of WISP-2 in hip joint cartilage of patients with osteoarthritis was significantly increased.Their research showed that WISP-2 was involved in cartilage dysplasia and cartilage abnormality.Peroxisome proliferator activated receptor ?(PPAR ?)is a ligand activated transcription factor,belonging to nuclear receptor superfamily.PPAR ? can regulate lipid metabolism and apoptosis,and inhibit inflammation.It was found that WISP-2 could inhibit the expression and activation of PPAR ? in 3T3L1 preadipocytes and NIH3T3 fibroblasts.We speculate that WISP-2 may play a role in promoting apoptosis by inhibiting PPAR ? expression and activation in chondrocytes.Many studies and clinical reports show that traditional swaddling still exists in many cultures.Complete extension and wrapping of the lower extremities can result in subluxation and dislocation of the hip.The apoptosis of chondrocytes in acetabulum and proximal femur of neonatal rat model was observed,and the expression of WISP-2 and PPAR ? in the model was measured.Then,in the primary chondrocytes cultured in vitro,we also studied whether WISP-2 might play an apoptosis promoting role in chondrocytes through PPAR ? gene overexpression or knockdown.This study provides a new way to understand the mechanism of DDH.Materials and Method:1 Materials1.1 AnimalsNewborn Wistar rats,provided by Changsheng biotechnology.Animal experiments were conducted in accordance with the guidelines for the care and use of experimental animals.1.2 ReagentsDulbecco's modified eagle's medium(DMEM)was purchased in hyclone(South Logan,UT,USA).Advanced glycation end products(AGE)-BSA was purchased from biovision(2221,Milpitas,CA,USA).PPAR ?(16643-1-ap),antibody was purchased from Proteintech(Wuhan).WISP-2(a7456,a12541)antibody was purchased from abclone(Wuhan)and Hoechst Staining Kit(C0003)from beyotime(Shanghai).TUNEL Kit(wl029a)and apoptosis detection kit(wla002a)were purchased from wanleibio(Shenyang).Safranin O was purchased from Solarbio(g2540,Beijing).3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-h-tetrazoliumbromide(MTT)was purchased in sigma(St.Louis,Mo,USA).Surgical medical tape(3m durapore,St.Paul,Minnesota).2Methods:2.1 The establishment of animal model and tissue collectionAs we have reported before,DDH model was established in Wistar newborn rats.DDH model was established by fixing the hind leg with medical tape in adduction and extension position of hip joint for 2 and 8 days.The newborn rats in the control group were fed normally without special treatment.On the 2nd and 8th day after fixation,the rats were euthanized randomly(200 mg / kg pentobarbital sodium).The acetabulum and femoral head were cut out completely and the follow-up experiment was carried out.2.2 Microarray analysisThe gene expression profiles of acetabular cartilage in DDH group and healthy control group were analyzed,and the experiment was carried out in Shanghai biotechnology company.After the feature extraction software 10.7(agent technologies)extracts the data,the original data is normalized by Quantile algorithm and Gene Spring Software 12.6.1.Differentially expressed gene was identifiedthrough fold change and the P value as calculated using the ttest and gene with a fold change(FC)?±2 differential expressionand a P value <.05 was considered.2.3 Safranine O stainingAfter fixation with 4% paraformaldehyde,the acetabulum and femoral head were embedded in paraffin and cut into 5 ? m sections.The sections were dewaxed,hydrated,stained with hematoxylin solution for 2 minutes,and then stained with safranine o for 2 minutes.The load-bearing areas of acetabulum top and femoral head were observed.The images were taken under microscope with 200 times magnification.2.4 Primary culture of chondrocytes andinfection with lentivirusChondrocytes were isolated from the acetabular cartilage and the epiphysis of the femoral head of neonatal rats.They were cultured in a humidified environment at 37 ? with 5% CO2.The cultured chondrocytes(3rd generation)were harvested and inoculated into the 6-well plate.In order to up regulate and down regulate the expression of WISP-2 or PPAR ?,chondrocytes were transfected with lentivirus,carrying plasmids of WISP-2 or PPAR ? overexpression(OE)or knockdown(sh RNA).72 hours later,the cells were collected for subsequent experiments.In order to investigate the effect of WISP-2 on apoptosis,chondrocytes transfected with WISP-2sh RNAs were treated with AGE of 200 ? g / ml 72 hours after infection to induce apoptosis.2.5 Real-time quantitative PCR(RT-q PCR)The total RNA was extracted from chondrocytes and transcribed into c DNA.The primer sequence of RT-q PCR is shown in Table 1.Theamplification conditions were: 94°C for 5 minutes;followedby 40 cycles of 94°C for 10 seconds,60°C for 20 seconds and72°C for 30 seconds;then incubated at 72°C for 2.5 minutesand 40°C for 1.5 minutes;melted from 60 ° C to 94 ° C,1 ° C/s.The m RNA level was normalized to ?-actin and the relativem RNA level was calculated using 2-??Ct method.2.6 Western blotWestern blot was used to detect the protein levels of WISP-2,PPAR ?,zfp423 and apoptosis related proteins.The total protein was extracted by RIPA,and the protein concentration was determined by BCA protein assay kit.The equivalent protein was separated by gel electrophoresis and the target band was detected by ECL substrate.The optical density of the band is analyzed by the gel image processing system(Gel-Pro-Analyzer software).2.7 TUNEL;Immunofluorescence;MTT;(annexin V &PI)double staining;Hoechst staining.These experiments are carried out according to the conventional experimental methods,and there are detailed steps in this paper.Results:3.1 microarrayA total of 246 highly regulated genes were found.Eight out of 104 up-regulated genes and eight out of 142 down-regulated genes were included in the study.The early expression changes of WISP-2 and PPAR ? in DDH suggest that WISP-2 and PPAR ? may be important factors for DDH initiation.3.2 Safranine O staining,gross measurement and detectionIn DDH group,the size of acetabulum and femoral head decreased significantly on the 8th day,which was manifested as shallow acetabulum,shorter long axis and short axis of acetabulum and femoral head.In addition,safranine O staining showed the degeneration of acetabulum and femoral head cartilage,and the apoptosis increased significantly.RT-q PCR was used to detect the relative m RNA levels of WISP-2 and PPAR ?.It was found that WISP-2 significantly increased and PPAR ? decreased in the femoral head and acetabulum on the 2nd and 8th day.3.3 Overexpression of WISP-2 induces chondrocyte apoptosisChondrocytes were isolated from acetabular cartilage and identified by immunofluorescence with CD44 antibody and type II collagen antibody.We detected the m RNA expression levels of chondrocyte markers,adipocyte markers and fibrocyte markers.The results showed that lentivirus infection alone did not affect the apoptosis of primary chondrocytes.After transfection,the level of WISP-2 was up-regulated at mRNA and protein levels.The overexpression of WISP-2 significantly reduced the activity of chondrocytes and increased the proportion of apoptotic cells.The levels of cleaved Cleaved caspase-3,cleaved Cleaved caspase-9,cleaved PARP and Bax increased significantly.3.4 Inhibition of AGE induced apoptosis by interference of WISP-2After transfection with WISP-2 sh RNA,the m RNA and protein levels of WISP-2 were also significantly reduced.The silencing of WISP-2 enhanced the decreased cell viability due to AGE and reduced the apoptosis of chondrocytes.By silencing WISP-2,the increased level of PARP caused by age was reduced.WISP-2 regulates PPAR ? expression in chondrocyte apoptosisWhen WISP-2 was overexpressed,the protein level of PPAR ? decreased significantly.After AGE treatment,the level of PPAR ? in chondrocytes decreased significantly.After WISP-2 was silenced,the level of PPAR ? partially recovered,the number of PPAR ? positive cells decreased,and the number of PPAR ? in the nucleus increased.3.5 PPAR ? attenuates the effect of WISP-2 on chondrocyte viability and apoptosisAfter transfection,PPAR ? m RNA and protein levels decreased in PPAR ? knockout cells,and PPAR ? m RNA and protein levels increased in PPAR ? overexpression cells.PPAR ? knockout can obviously inhibit the activity of chondrocytes and promote the apoptosis of chondrocytes.The overexpression of PPAR ? decreased the apoptosis of chondrocytes induced by WISP-2 overexpression,enhanced the decreased cell viability and increased the protein levels of PARP and Bax.WISP-2 is mainly expressed in the cytoplasm of chondrocytes,which is negatively correlated with Zfp423,the key transcriptional activator of PPAR ?.Conclusions : The acetabulum and femoral head of DDH model rats are underdeveloped,and apoptosis is increased,the expression of WISP-2 is up-regulated in the cartilage of hip joint of DDH model rats,and the expression of PPAR ? is down regulated.In cell culture,overexpression of WISP-2 can lead to over apoptosis of chondrocytes and inhibit PPAR ? expression,while interference with WISP-2 expression can reduce AGE induced apoptosis and increase PPAR ? expression.Overexpression of PPAR ? can also reduce WISP-2-induced apoptosis.That is to say,WISP-2 may induce chondrocyte apoptosis by regulating PPAR ? expression,which may be one of the reasons for DDH activation.