The Study Of The Mechanism Of The Piezo1 Protein-induced Apoptosis Of The Ostearthritis Chondrocytes Through The MAPK Signal Pathway

OBJECTIVE To study whether the new Mechanically-activated cation channel Piezo1 protein can cause the apoptosis of the human chondrocytes under compressive loading,using a Flexercell unit by activating MAPK signal pathywayMETHODS Primary human chondrocytes were isolated,cultured,and then subjected to the static compressive loading for 0h,2h,12 h,24h and 48 h,respectively.As well as the inhibitor of the Piezo1,Gs MTx4,the inhibitor of the ERK5,BIX02188,and the inhibitor of the ERK1/2,PD98059.The immunofluorescence was used to locate the expression of the Piezo1 protein the ERK5,and the ERK1/2.The expressions of Piezo1 and the MAPK/ERK5 and the MAPK/ERK1/2 were assessed by reverse transcription-polymerase chain reaction(RT-PCR)and the Western-blot,as well as the apoptosis gene B cell lymphoma/leukemia-2(Bcl-2),Bel-associated X protein(Bax),Caspase-3 and BAD.In addition,Piezo1 inhibitor,Gs MTx4,was used to block the MA cation channel Piezo1,served as a positive control.Annexin V-PI was used to detect the apoptosis of the OA chondrocytes.RESULTS(1)The location of the Piezo1 was expressed in the nucleus and cytoplasm of the chondrocytes.While the expressed of the ERK5 and the ERK1/2 was proved in the cytoplasm of the cells.(2)The expression of the Piezo1、ERK5、ERK1/2、BAD、and Bax m RNA tested by the RT-PCR in the OA chondrocytes is weak,however,the 2h stretch force group was significant increased(P<0.05),the 24 h stretch force group was the highest expression while the expression of the 48 h group was decreasing.However,the expression of the Bcl-2 in the 12 h group was decreasing and the 12 h stretch force group was the lowest expression while the 24 h stretch force group was increasing.In the Gs MTx4 group the expression of the Piezo1、ERK5、ERK1/2、BAD、Bax was decreasing while the Bcl-2 was increasing.In the BIX02188 group,the expression of the ERK5、BAD 、 Bax was increasing while the Bcl-2 was decreasing,but the expression of the Piezo1 was not change.As well as the MAPK/ERK/1/2.The result of the Western-Blot had shown that the expression of the Piezo1 protein in the 2h stretch group was higher than the 0h group(P<0.05),while the expression of the 48 h group was lower than the 24 h group(q<0.001,P<0.05).(3)The result of AV-PI had shown that the 2h stretch forcegroup had increased early stage of apoptosis.The 12 h stretch force group had increased late stage of apoptosis,and the 24 h stretch force group’s apoptotic rate was the highest,while the apoptotic rate of the 48 h group was lower than the 24 h stretch force group(q<0.05,P<0.05).The Gs MTx4 could inhibit the late stage of apoptosis.(4)The result of the RT-PCR and the Western-Blot had the same trend as well as the result of the AV-PI.CONCLUSION Piezo1 plays an important role in the apoptosis of the human chondrocyte through the MAPK/ERK5 and MAPK/ERK1/2 signal pathway.