Molecular Mechanism Of ShRNA-TRPV4 Regulating Apoptosis Of Chondrocytes Under Mechanical Stress In Vitro

Osteoarthritis(OA)is a disease closely related to human biomechanics.Commonly seen in elderly women,especially in manual workers,the main symptoms of knee pain and limited movement.For patients with severe osteoarthritis of the knee,there is usually a change in the biological force line of the lower knee joint.At the cellular level,the pathogenesis of osteoarthritis is cartilage matrix degradation and chondrocyte apoptosis.Previous studies have focused on the degradation of cartilage matrix,the apoptosis of chondrocytes and the invasive changes of synovitis in the articular cavity of the knee joint.However,there are few studies on the biosignals of mechanical distraction stress of knee chondrocytes caused by the changes of lower limb force lines.In this study,the mechanical stretching stress model of chondrocytes in vitro was constructed by Flexcell mechanical stretching apparatus.According to the results of pre-experiment,the experimental group was divided into 0-hour mechanical stretching stress group,6-hour mechanical stretching stress group,24-hour mechanical stretching stress group,36-hour mechanical stretching stress group and 72-hour mechanical stretching stress group.And then explore the effect of Biomechanics on chondrocytes.In order to promote the excessive apoptosis of chondrocytes under mechanical stretching stress,mechanical activated ion channel TRPV4 was used as the second messenger and ERK5/MAPK signaling Pathway was used to activate the expression of apoptotic genes.The ShRNA-TRPV4 interfering plasmid was constructed by ShRNA interfering technique,and the gene of TRPV4 was silenced.Lentivirus was used as the vector of ShRNA-TRPV4 interfering plasmid to explore the protective mechanism of ShRNA-TRP4 on chondrocyte apoptosis induced by mechanical stretching stress.The results showed that ShRNA-TRPV4 could protect chondrocytes from apoptosis by blocking the expression of mechanical activated ion channel TRPV4,decreasing the calcium content,blocking the expression of ERK5/MAPK signaling pathway,and then blocking the expression of apoptotic genes.Part ?:Construction and screening of lentiviral vector targeting ShRNA interference vector of TRPV4 geneObjective:To construct ShRNA-TRPV4 interference plasmid by ShRNA interference technique and screen the ShRNA-TRPV4 interference sequence using lentivirus as transfection vector.Methods:Human renal epithelial cell line 293T was used as the research object.The ShRNA sequence was constructed by using lentivirus as the transfection vector and LV3 plasmid as the vector.Three ShRNA-TRPV4 sequences were constructed:LV3-TRPV4-homo-2899,LV3-TRPV4-homo-3207 and LV3-TRPV4-homo-4108.Lentivirus transfection technology was used to transfect ShRNA-TRPV4 into human renal ePithelial cell line 293T cells.The transfection efficiency of lentivirus was observed by inverted fluorescence microscoPe.The expression of ShRNA interference sequence was detected by RT-PCR and Western Blot.Results:(1)According to Designer 3.0(GenePharma)software,three ShRNA TRPV4 interference sequences were designed for TRPV4 gene sequence.(2)The Plasmid pLV3-ShRNA-ZsGreen-TRPV4 was digested by XhoI and linearized by agarose gel electroPhoresis.The results of enzyme digestion showed that hTRPV4-21 was a Positive clone.(3)Using 293 T cells as target cells,10-1,10-2,10-3,10-4 viruses were transfected to select the best viral titer.Obviously,the transfection efficiency was the highest when MOI=50.(4)RT-PCR results showed that TRPV4-homo-3207 sequence group,TRPV4-homo-4108 sequence group and negative control group were significantly different(F?18.219,P?0.000<0.001);and TRPV4-homo-3207 sequence group compared with the other three groups,TRPV4-homo-3207 relative expression decreased with statistical significance differences(q=2.221,P=0.0192<0.05).(5)Western-Blot analysis showed that the relative expression of TRPV4/beta-actin Protein in TRPV4-homo-3207 sequence group,TRPV4-homo-4108 sequence group and negative control group was significantly different(F=21.327,P=0.000<0.001),and TRPV4-homo-3207 sequence group was significantly different from the other three groups(F=-21.327,P=0.000<0.001).The relative expression of mRNA decreased with statistical difference(q=1.872,P=0.0215<0.05).Conclusion:RNA interference can play a role in gene silencing.TRPV4-homo-3207 sequence can effectively silence the expression of TRPV4 gene in 293T cells by lentiviral transfection,and can be used as an effective interference sequence.Part ?:To explore the mechanism of ShRNA-TRPV4 inhibiting excessive apoptosis of chondrocytes under mechanical stretch stress through ERK5/MAPK signaling pathway.Objective:To explore the mechanism of ShRNA-TRPV4 inhibiting excessive apoptosis of chondrocytes induced by mechanical stretch through ERK5/MAPK signaling pathway.Methods:Human chondrocytes were obtained from the cartilage tissue of total knee arthroplasty(TKA)in our hospital.The Primary chondrocytes were identified by immunohistochemical method.Flexcell-4000T system was used to construct an in vitro mechanical distraction stress chondrocyte model.According to the intervention factors,the distraction amplitude was set at 20%,the degeneration and recovery cycles were 6 cycles per minute,and the interval of each deformation was 10 seconds.According to the treatment of mechanical stretch stress,ShRNA-TRPV4 lentivirus transfection and ERK5 inhibitor BIX02188 divided chondrocytes into the following groups:0 h mechanical stretch stress group,6 h mechanical stretch stress group,24 h mechanical stretch stress group,36 h mechanical stretch stress group,72 h mechanical stretch stress group.After ShRNA-TRPV4 lentivirus transfection,the mice were divided into four groups:0 h mechanical stretching stress?ShRNA-TRPV4 group,6 h mechanical stretching stress?ShRNA-TRPV4 group,24 h mechanical stretching stress+ ShRNA-TRPV4 group,36 h mechanical stretching stress+ShRNA-TRPV4 group,72 h mechanical stretching stress+ShRNA-TRPV4 group;after treatment with ERK5 inhibitor BIX02188,the mice were divided into 0 h mechanical stretching stress+ShRNA-TRPV4 group.Tensile stress+BIX02188 group,6 h mechanical stretching stress+BIX02188 group,24 h mechanical stretching stress+BIX02188 group,36 h mechanical stretching stress+BIX02188 group,72 h mechanical stretching stress+BIX02188 group.The chondrocytes were transfected with ShRNA-TRPV4 lentiviral vector constructed in the first part.The expression of TRPV4 Protein and ERK5 Protein was localized by immunofluorescence staining.The expression of TRPV4 Protein and ERK5 Protein was detected after mechanical stretch stress treatment.The relative expression levels of mRNA in TRPV4,Caspase-3,Caspase-9 and Caspase-12 were detected by real-time PCR.Western-Blot detected the protein expression levels of TRPV4 and ERK5.Fluo-3 and AM kit were used to detect intracellular calcium content.The apoptosis rate of each group was detected by flow cytometry and Hoechst staining.Results(1)Chondrocyte morphology showed that the morPhology of the third generation chondrocyte was in the best state,so the third generation chondrocyte was selected as the target cell of the following study.(2)Toluidine blue specifically binds to the polysaccharides in the cytoplasm of chondrocytes,thus making the cytoplasm of chondrocytes purple red,thus verifying the cell characteristics of seed cells,and preparing for the following experiments.(3)The Aggrecan immunohistochemical staining of P3 chondrocytes showed that the intracytoplasmic brown staining was strong and Aggrecan expression was high in P3 chondrocytes.The immunohistochemical staining of collagen type II showed that the intracytoplasmic brown staining was strong and the expression was high.(4)The chondrocytes were transfected with ShRNA-TRPV4 lentivirus,and the visual field cells were stained with GFP green fluorescent Protein under fluorescence.The transfection efficiency of lentiviruses 48 hours after lentiviral transfection was(52.6±2.9)%and 72 hours after lentiviral transfection was(87.4±11.4)%.(5)Morphological analysis by inverted optical microscope:the chondrocytes in the 6 h stretch stress control group shrank into apoptotic bodies only sporadically distributed in the visual field,and the proportion of chondrocytes shrank into apoptotic bodies in the 24 h stretch stress control group was higher than that in the 6 h stretch stress control group.The proportion of chondrocyte apoptosis corpuscles in 36 h group was lower than that in 24 h group,and that in 72 h group was lower than that in 36 h group.There was no significant difference between 0 h mechanical stretch stress+ShRNA-TRPV4 group and 0 h stretch stress control group.24 h mechanical stretch stress+ShRNA-TRPV4 group compared with 24 h stretch stress control group,the proportion of apoptotic bodies decreased.The chondrocytes of 36 h and 72 h mechanical stretch stress+ShRNA-TRPV4 group had the same expression characteristics as those of 36 h and 72 h stretch stress control group.0 the mechanical tensile stress+BIX02188 group had the same expression characteristics.(6)Immunofluorescence staining localized the expression of TRPV4 and ERK5 Protein in chondrocytes.It was found that TRPV4 Protein was exPressed in the cytoPlasm of chondrocytes.The expression of TRPV4 Protein in 24 h mechanical stretching stress group was significantly higher than that in other mechanical stretching stress groups.ERK5 Protein was expressed in the cytoplasm of chondrocytes.With the extension of loading time of mechanical stretching stress,the expression of ERK5 protein in 24 h mechanical stretching stress group was significantly higher than that in other mechanical stretching stress groups.(7)The relative expression of TRPV4 mRNA in 24 h mechanical stretching stress group was significantly higher than that in 6 h mechanical stretching stress group(P<0.05)and 0 h mechanical stretching stress group(P<0.05);ERK5,Caspase-3,Bcl-2,BAD and Bax had the same expression characteristics.(8)Fluo-3,AM kit was used to detect the intracellular calcium content in the chondrocytes of the stress control group and the stress+ShRNA-TRPV4 group.The results showed that the fluorescence intensity of calcium ion in the 24 h mechanical stretching stress group was significantly higher than that in the 6 h mechanical stretching stress group(P<0.05)and the 0 h mechanical stretching stress group(P<0.05).The fluorescence intensity of calcium ion in chondrocytes of the force group was significantly higher than that of the mechanical stretching stress group at 36 h(P<0.05)and the mechanical stretching stress group at 72 h(P<0.05).(9)Western Blot assay showed that the relative expression of TRPV4 Protein in 24 h mechanical stretching stress group was significantly higher than that in 6 h mechanical stretching stress group(P<0.05)and 0 h mechanical stretching stress group(P<0.05).However,the relative expression of TRPV4 Protein in stress+ShRNA-TRPV4 group was significantly lower than that in stretch stress control group(P<0.05).(10)AV-PI kit and Hoechst staining were used to detect the apoptosis of chondrocytes in the mechanical stretching stress control group.The apoptosis rate of chondrocytes in the mechanical stretching stress group at 24 h was significantly higher than that in the mechanical stretching stress group at 6 h(P<0.05)and the mechanical stretching stress group at 0 h(P<0.05).The apoptosis rate of chondrocytes in the mechanical stretching stress group was significantly higher than that in the mechanical stretching stress group at 36 h(P<0.05)and the mechanical stretching stress group at 72 h(P<0.05).Conclusion:ShRNA-TRPV4 can inhibit the expression of TRPV4 by blocking the expression of TRPV4,decrease the content of calcium ion,and block the expression of ERK5/MAPK signaling pathway in stretch-induced stress cell model,thereby blocking the expression of apoptotic genes,which Plays a protective role in chondrocyte apoptosis.