The Role Of 17β-estradiol On Acid-induced Rat Articular Chondrocytes Apoptosis And Its Mechanisms

Rheumatoid arthritis is a chronic immune syndrome mainly characterized by synovial inflammation,cartilage erosion and joint destruction.At present,great progress has made to treat RA.It is controlled by first-line non-steroidal anti-inflammatory drugs combined with anti-rheumatic drugs.The disease,although the inflammation has been successfully controlled,still cannot prevent the progression of RA,and the curative effect is still not satisfactory.Therefore,actively searching for drugs to protect cartilage from damage and joints from damage has become the common expectation of scholars at home and abroad.Epidemiological evidence suggests that the etiology and pathogenesis of rheumatoid arthritis(RA)are closely associated with estrogen metabolism and deficiency.Articular damage was reduced by estrogen pretreatment.Estradiol replacement therapy ameliorates local inflammation and knee joint swelling in ovariectomized models of RA.The mechanistic basis for the protective role of17β-estradiol(17β-E2)is poorly understood.Acid-sensing ion channel 1a(ASIC1a),as a sodium-permeable channel,plays a pivotal role in acid-induced rat articular chondrocytes injury.The aims of this study were to evaluate the role of 17β-E2 in acid-induced chondrocytes injury and to determine the effect of 17β-E2 on the level and activity of ASIC1 a protein.1.The role of 17β-E2 in acid-induced injury of articular chondrocytesIn vitro experiments,we explore the effects of 17β-E2 on the growth rate of articular chondrocytes in rats under normal conditions and the survival rate of chondrocytes,changes in cell morphology and mitochondrial membrane potential under acid-induced pathological conditions.Normal rat articular chondrocytes were divided into control group,500,1000,2000 n M 17β-E2 group,48 h after 17β-E2 treatment,MTT detection demonstrated that 17β-E2 had no significant effect on the survival rate of rat articular chondrocytes;Further,the survival rate of rat articular chondrocytes was explored after pretreatment by estradiol and acidification,normal chondrocytes were grouped as follows: [1] Control,p H 6.0,p H 6.0+500/ 1000/ 2000 n M 17β-E2 group,after 48 h of estradiol stimulation and acidification were performed at p H 6.0 for 3 h,the method of MTT was used to measure cell viability;[2] Control,p H 6.0,p H 6.0+17β-E2,p H 6.0+ICI,p H 6.0+ICI+17β-E2,after pretreatment with estrogen receptor blocker ICI and17β-E2,acidified at p H 6.0 for 3 h,using MTT assay to explore cell viability;[3]Control,p H6.0,p H6.0+17β-E2,p H6.0+Pc TX1,p H6.0+Pc TX1+17β-E2 group,pretreatment with ASIC1 a specific blocker Pc TX1 or 17β-E2,acidification for 3 hours under acidic conditions at p H 6.0,the assay of MTT was employed to measure cell viability.At p H 6.0,17β-E2 can significantly alleviate acid-induced chondrocytes survival rate;Inhibitory of estrogen receptors with ICI187280 reverses the estradiol-mediated protective effect;and the use of ASIC1a’s specific blocker Pc TX1 will have the same protective effect as 17β-E2,but after the pretreatment of Pc TX1,the further treatment of 17β-E2 did not produce an additional protection,which indicates that the activation of ASIC1 a may be involved 17β-E2-mediated cartilage protection;Cell morphology and Rhodamine123 staining was used to analyze acid-mediated cartilage protection,and it was found that 17β-E2 can significantly improve the morphology and disruption of mitochondrial integrity of chondrocytes caused by acidification.In summary,these functional tests indicate that 17β-E2 reducesacid-induced cartilage damage.2.The role of 17β-E2 on ASIC1 a protein expression and activity of articular chondrocytes Rat articular chondrocytes were treated with 17β-E2 at different concentrations for 72 h.ASIC1 a protein level and gene transcription were measured by Western blotting and Q-PCR,respectively.The best concentration of 17β-E2-reduced ASIC1 a protein expression was determined.Chondrocytes were incubated with 1000 n M 17β-E2 for different times(24,48,and 72 h),respectively,the protein expression and gene transcription of ASIC1 a were tested by Western blotting and Q-PCR.After 72 h of incubation at the optimal concentration of 1000 n M,Immunofluorescence and laser confocal technology were used to detect ASIC1 a level.The effects of 17β-E2 on ASIC1 a protein expression and activity were explored separately.These results together confirmed that 17β-E2 can reduce the protein expression and activity of ASIC1 a,but has no effect on ASIC1 a gene transcription.3.Mechanisms of 17β-E2 in attenuation of ASIC1 a protein expression of articular chondrocytes Combined protein synthesis inhibitor CHX and 17β-E2 to incubate chondrocytes for a certain period of time,the protein expression of ASIC1 a was significantly lower than the CHX group,which preliminarily demonstrated that 17β-E2 promotes the protein degradation of ASIC1a;further use of proteasome inhibition MG-312 and calpain inhibitor ALLN,there was no effect on the 17β-E2-mediated reduction of ASIC1 a protein.After using the lysosomal CQ and the autophagy inhibitor 3-MA to block the autophagy lysosomal pathway.It can obviously reverse the 17β-E2-induced degradation of ASIC1 a protein,which further illustrates that 17β-E2 promotes the degradation of ASIC1 a by the autophagy lysosomal pathway;At the same time,the results also showthat 17β-E2 increases Beclin-1 and LC3-Ⅱ protein expression,and after CQ pretreatment,the expression of Beclin-1 and LC3-Ⅱ significantly increased compared to the 17β-E2 group,and immunofluorescence also confirmed this phenomenon;These results indicate that 17β-E2 activates autophagy and autophagy lysosomal pathway take part in 17β-E2-induced ASIC1 a protein degradation;4.Role of estrogen receptors in 17β-E2-mediated ASIC1 a protein levels in articular chondrocytes Primary chondrocytes were divided into control group,17β-E2,blocker of estrogen receptor with ICI,ICI+17β-E2 group,Western blot detected ASIC1 a protein expression,result showed that the function of 17β-E2 on ASIC1 a level was blocked by ICI;After pretreatment with special antagonists MPP/ PHTPP/ G15(ERα,ERβ and GPER),result showed that the attenuation of ASIC1 a protein expression induced by 17β-E2 was reversed by ERα blocker MPP when compared to the other two antagonists.In addition,silencing of ERα gene also significantly increased ASIC1 a protein levels compared to17β-E2.These results indicated that ERα,but not ERβ or GPR30,is involved in17β-E2-induced ASIC1 a protein degradation.5.The role of 17β-E2 in acid-induced apoptosis in rat articular chondrocytes Control,p H6.0,p H6.0+17β-E2,and p H6.0+Pc TX1 were grouped.After a certain pretreatment,chondrocytes apoptosis rate and morphological changes were tested after acidification by Flow cytometry and Hoechst staining.To further evaluate the effects of17β-E2 on chondrocytes apoptosis,cells were cultured with ERs blocker ICI,the inhibitory function of 17β-E2 on Caspase-9 and PARP level were reversed.The results indicate that 17β-E2 attenuates acid-induced chondrocytes apoptosis;We found that Pc TX1,a special inhibitor of ASIC1 a,has a better inhibitory effect on apoptosis,indicating that ASIC1 a activation is linked to chondrocytes apoptosis induced by acid.To further confirm the relationship between 17β-E2-mediated protection and ASIC1a-induced chondrocytes apoptosis,compared with Pc TX1,the combined treatment with Pc TX1 and 17β-E2 did not change the level of ASIC1 a,Caspase-9 or PARP.These data shown that 17β-E2 prevents acid-induced chondrocytes apoptosis through ASIC1 a channel.Taken together,these results indicate that 17β-E2 reduces ASIC1 a levels and is responsible for acid-mediated chondrocytes protection.Results showed that pretreatment with 17β-E2 attenuated acid-induced damage,suppressed apoptosis,and restored mitochondrial function.Further,17β-E2 was shown to reduce protein levels of ASIC1 a through the autophagy-lysosomal pathway,to protect chondrocytes from acid-induced apoptosis,and to induce ASIC1 a protein degradation through the ERα receptor.Taken together,these results show that the use of 17β-E2 may be a novel strategy for the treatment of RA by reducing cartilage destruction through down-regulation of ASIC1 a protein levels.