The Mechanism Of Volume-sensitive LRRC8A Chloride Channel Regulating Type II Collagen Of Chondrocyte

Objective:To explore the expression of volume-sensitive LRRC8 A chloride channel in normal and OA chondrocytes,and to analyze the effect of the activation and expression level of LRRC8 A ion channel on the expression of type II collagen,for providing a new experimental basis of development of chloride channel type anti-osteoarthritis drugs.Methods:Relatively normal chondrocytes and ones from OA tissue were isolated from the patients' femoral head and knee joints after operation,and toluidine blue staining was used to preliminarily identify whether the cultured cells were chondrocytes.The m RNA levels of COL II and LRRC8 A chloride channels in chondrocytes of femoral neck fracture patients and osteoarthritis patients were compared by q PCR and immunofluorescence experiments.After treating chondrocytes with chloride channel blockers NPPB,LRRC8A-si RNA and different osmotic pressure media,the m RNA levels of COL II and LRRC8 A genes were quantified.Results:The results of toluidine blue staining of chondrocytes obtained from patients were positive,and the cells we cultured were preliminarily identified as chondrocytes.The m RNA expression levels of COL II and LRRC8 A of chondrocytes from patients with femoral neck fracture were(1.004 ±0.082)and(1.002±0.057).The m RNA levels of COL II and LRRC8 A in OA patients' chondrocytes were(0.27±0.06)and(1.998±0.141)times of the control group,respectively.The m RNA expression of COL II gene in chondrocytes of OA patients decreased,and the expression of LRRC8 A gene increased(P <0.05),and the difference between the groups was statistically significant.In the immunofluorescence experiment,the average fluorescence intensity of COL II in normal chondrocytes was(25.877±3.034)A.U,and(44.663 ± 3.493)A.U in OA group.The average value in control group was 0.579 times that of OA group.The average fluorescence intensity of LRRC8 A chloride channel in normal chondrocytes was(19.16±1.934)A.U,and(54.756±4.772)A.U in OA group,which was2.858 times that of normal chondrocytes.In the experiment of blocking chloride channel by NPPB,the COL II m RNA expression of chondrocytes in the control group was(1.031 ±0.247),after blocking the chloride channel using NPPB with a working concentration of 100 nmol/L,the COL II gene m RNA expression in chondrocytes obviously decreased to(0.278±0.054)times of the control group,P <0.05,and the difference between the groups was statistically significant.Cultured In hypertonic culture medium(441 m Osm/L),the expression levels of COL II and LRRC8 A gene m RNA of chondrocytes were(1.006±0.017)and(1.022±0.202)respectively,and in hypotonic culture medium(160 m Osm/L),expression of COL II and LRRC8 A m RNA were respectively(0.519 ± 0.082)and(1.77 ± 0.117)times of hypertonic group.In addition,the m RNA levels of COL II and LRRC8 A gene of isotonic group(300 m Osm/L)were respectively.(0.76±0.037)times and(1.867 ± 0.319)times of hypertonic group,P <0.05,the difference between the groups was statistically significant.In the LRRC8A-si RNA transfection experiment,the mRNA expression levels of COL II and LRRC8 A genes in the control group were(1.053 ± 0.321)and(1.015 ± 0.182).After transfection with LRRC8A-si RNA at a working concentration of 100 nmol / L,the m RNA expression of COL II and LRRC8 A genes in chondrocytes were significantly reduced,which were(0.21±0.027)times and(0.031±0.004)times of the control group,P <0.05,the difference between the groups was statistically significant.The COL II mRNA expression of chondrocytes cultured in hypertonic culture medium(441 m Osm/L)was(0.963±0.06),hypotonic culture group(160 m Osm/L),hypertonic culture medium(441 m Osm/L)+ NPPB(100 nmol/L)group and hypotonic culture medium(160m Osm/L)+ NPPB(100 nmol/L)group,the expression of COL II gene m RNA all decreased,which were(0.637±0.09)times,(0.495±0.092)times,and(0.149±0.137)times of the gene level of hypertonic group.Among these groups,The expression of COL II gene m RNA in hypotonic culture medium(160 m Osm/L)+ NPPB(100 nmol/L)group was less significant than that in the first two groups,P <0.05,and the difference between the groups was statistically significant.Conclusion:1.Compared with the high infiltration(441 mOsm/L)pressure in the joint,the expression of type II collagen in normal chondrocytes cultured with isoosmosis(300 m Osm/L)and hypoosmosis(160 m Osm/L)decreased significantly.The hyperosmosis(441 m Osm/L)culture conditions may have the function of protecting type II collagen in normal chondrocytes.2.Down-regulation of the LRRC8 A chloride ion channel can inhibit the expression of type II collagen in normal chondrocytes.3.Inhibition of the LRRC8 A chloride ion channel further promoted the catabolism of type II collagen in normal chondrocytes at low osmotic pressure(160 m Osm/L).