The Mechanism Of The The GPR30 Receptor In Osteoarthritis Treatment Through Inhibition Of Ferroptosis And Synovial Inflammation

Background and objectiveOsteoarthritis（OA）is the most common joint disease,and OA causes joint pain,decreased mobility and loss of function,ultimately leading to a restricted lifestyle and reduced quality of life for patients.OA is characterized by the progressive articular cartilage degradation,synovial inflammation and subchondral bone changes,leading to joint destruction.Epidemiological studies have reported that 18%of women over the age of 60 have OA and that postmenopausal women have a rapidly increasing risk of developing OA compared to premenopausal women and men at the same age.It is therefore generally accepted that the postmenopausal decline in estrogen levels is a key factor in the development of OA.There are many causes of osteoarthritis,including chondrocyte senescence,death and inflammation.However,recent studies suggest that ferroptosis may play an important role.ferroptosis is a newly identified type of programmed cell death induced by excess iron ions and reactive oxygen species（ROS）and driven by lipid peroxidation.Studies have shown that osteoarthritis shares some common features with ferroptosis,such as abnormal iron metabolism,lipid peroxidation and mitochondrial dysfunction.Iron levels are significantly elevated in the elderly,and this change is more pronounced in older women,a phenomenon that may be related to estrogen deficiency in postmenopausal older women.Thus,the relatively higher susceptibility of older women to OA may be due to ferroptosis from iron overload and is closely related to estrogen levels.The inflammatory response that occurs in the joints and periarticular tissues is one of the main factors that accelerates the progression of OA.Studies have shown that synovial tissue is activated during the pathogenesis of osteoarthritis,releasing a variety of inflammatory response proteins that exacerbate the destruction of the cartilage matrix.Estrogen is an important regulator of chondrocyte function;it increases the synthesis of cartilage matrix-specific components and decreases the expression of inflammatory and catabolic genes,and estrogen is also an important regulator of the immune response,which can inhibit inflammatory responses.However,estrogen replacement therapy increases the risk of reproductive malignancies in female patients,limiting its use in clinical practice.Articular cartilage is an estrogen-sensitive tissue that expresses the classical nuclear estrogen receptors ERαand ERβ,as well as the membrane estrogen receptor,GPR30,which binds to estrogen and mediates a rapid non-genomic and genomic transcriptional response to estrogen,bypassing the side effects of estrogen.The protective roles of GPR30 in degenerative and inflammatory diseases have been extensively studied.However,despite the widespread expression of GPR30 in joint tissues,few studies have investigated its effects on skeletal and joint health.The preventive and therapeutic effects of GPR30 in OA are unclear,and its role in chondrocyte ferroptosis and synovial inflammation is unknown.Therefore,this study investigated the role and mechanism of GPR30 in OA to clarify its potential as a therapeutic target for OA.1.Statistical analysis of genome-wide RNA sequences from cartilage tissues of osteoarthritis patients and normal cartilage tissues（GEO No.GSE114007）was performed to compare the differences in transcript levels of the GPR30 gene between cartilage tissues of osteoarthritis patients and normal cartilage tissues.2.Cartilage tissues from osteoarthritis patients with stage III or higher joint replacement were collected and the difference in GPR30 protein expression between severely damaged and undamaged cartilage tissues was analyzed by immunohistochemical staining and Western blot.3.GPR30 knockout mice were generated using CRISPR-Cas9 technology and the DMM mouse model of OA was established to investigate the effect of GPR30 deletion on the progression of OA by comparing the differences in postoperative behavioral scores and histopathological scores of joints in wild-type and GPR30 knockout mice with those in DMM.4.DMM mouse models of OA was established in wild-type mice,treated with or without the GPR30 agonist G1,to compare the effects on behavioral scores and joint histopathological scores,and to explore the effects of agonism of the GPR30 receptor on the progression of OA.5.Correlation of GPR30 expression with ferroptosis driver,marker and suppressor genes in cartilage tissue using whole genome RNA sequencing data.6.To analyze the differences in the expression of glutathione peroxidase（GPX4）and4-hydroxynonenal（4-HNE）between osteoarthritic cartilage tissue and normal cartilage tissue by immunohistochemical staining and Western blot,and to analyze the differences in Fe²⁺content between the joint fluids of patients with OA and normal joint fluids.To verify the role of ferroptosis in the pathogenesis of OA.7.The effects of GPR30 on chondrocyte activity,matrix anabolic capacity and ferroptosis related metrics were tested by simulating the ferroptosis process in human primary chondrocytes and ATDC5 chondrocytes using the ferroptosis agonist Erastin and administering E2,G1 and G15.8.Proteome sequencing of untreated,Erastin and Erastin+G1 treated chondrocytes.Investigate the protective mechanism of GPR30 against ferroptosis in chondrocytes.9.Changes in the expression of related proteins in chondrocytes after treatment with Erastin and G1 were detected by Western blot,and the cells were knocked down for GPR30,ferritin heavy chain 1（FTH1）and Yes-associated protein-1（YAP1）expression to observe the altered sensitivity of protein knockdown chondrocytes to ferroptosis inducers.10.The effect of G1 on the expression of FTH1 protein and 4-HNE in OA model mice was observed by immunohistochemical staining.11.Differences in the expression of GPR30 protein in synovial tissues from osteoarthritis patients and normal tissues were analyzed by immunohistochemical staining and Western blot.The effect of GPR30 deletion on synovial inflammation was explored by pathological staining analysis of differences in synovial inflammation scores and matrix-degrading enzyme expression in GPR30 knockout mice compared with wild-type mice after DMM surgery.12.DMM mouse models of OA was established in wild-type mice and treated with the GPR30 agonist G1 to investigate the effect of GPR30 receptor agonism on synovial inflammation by comparing the differences in synovial inflammation scores and matrix-degrading enzyme expression between the DMM mice and the G1-treated mice after DMM surgery.13.IL-1βwas used to establish a cell model in human primary synovial fibroblasts and G1 was induced,and changes in the expression of matrix degrading enzymes and inflammatory factors after G1 treatment were detected by enzyme-linked immunosorbent assay（ELISA）,Western blot and q RT-PCR.14.Synoviocytes were cultured in medium containing IL-1βor G1 for 24 h.After replacing the medium with new medium,human fibroblast synoviocytes were co-cultured with human primary chondrocytes using the Transwell assay and changes in the expression of markers of chondrocyte stromal catabolism and synthesis were detected by q RT-PCR and Western blot.Results1.GPR30 expression is reduced in osteoarthritic cartilage tissueStatistical analysis of whole-genome RNA sequencing revealed that the transcript level of the GPR30 gene was significantly decreased in osteoarthritic cartilage tissue compared to normal cartilage tissue.Immunohistochemical staining and Western blot analysis showed that the expression of GPR30 protein was significantly lower in severely damaged cartilage tissues than in undamaged cartilage tissues.2.GPR30 knockout exacerbates osteoarthritis phenotype after DMM surgery in miceUsing CRISPR cas9 technology to establish GPR30 knockout mice and DMM mouse models of OA,it was found that behavioral scores and joint histopathology scores were more significantly improved in GPR30 knockout OA mice compared to wild-type OA mice undergoing DMM surgery.3.GPR30 agonist G1 attenuates post-operative OA phenotype in mice with DMMMouse OA models of DMM established in wild-type mice showed that treatment with the GPR30 agonist G1 significantly improved behavioral scores and joint histopathology scores after DMM surgery in mice.4.Ferroptosis plays an important role in the OA process and is negatively correlated with the expression of GPR30Correlation analysis of whole-genome RNA sequencing data showed that GPR30expression in cartilage tissue was negatively correlated with ferroptosis response driver and marker genes and positively correlated with ferroptosis suppressor genes.Immunohistochemical staining and Western blot showed that the expression of glutathione peroxidase（GPX4）was significantly reduced in osteoarthritic cartilage tissue,whereas the expression of 4-hydroxynonenal（4-HNE）,a marker of ferroptosis,was significantly increased.It was also found that the Fe²⁺content in joint fluid and tissues of OA patients was significantly higher than that of the normal population.5.GPR30 agonist G1 attenuates effects of ferroptosis agonist Erastin on chondrocyte ferroptosis phenotypeUsing the ferroptosis agonist Erastin to mimic the ferroptosis process in human primary chondrocytes and ATDC5 chondrocytes,it was found that E2 and G1 could enhance cell viability in a concentration-dependent manner and that their protective effects could be inhibited by G15;whereas G1,at a concentration of only 1/10 that of E2,showed a similar protective effect as E2.G1 rescued the expression of COL2 and ACAN in Erastin-treated chondrocytes,significantly reduced intracellular levels of free ferrous ions,ROS,MDA and lipid peroxidation,and restored the level of mitochondrial membrane potential in chondrocytes as detected by Western blot.6.G1 reduces Erastin-induced cellular ferroptosis by increasing FTH1expression in chondrocytesProteome sequencing showed that G1 acts mainly by inhibiting chondrocyte ferroptosis and is associated with the Hippo pathway,with ferritin being the most significantly up-regulated protein in the ferroptosis pathway,consistent with the positive correlation between GPR30 expression and FTH1 in the GSE114007 dataset.Erastin treatment decreased FTH1 protein expression in chondrocytes,whereas G1 treatment significantly increased its expression as shown by Western blot.Knockdown of GPR30and FTH1 expression in chondrocytes resulted in increased sensitivity of chondrocytes to ferroptosis inducers,whereas the protective effect of G1 against ferroptosis in chondrocytes was significantly reduced.7.GPR30 regulates the transcription of FTH1 by increasing the expression level of YAP1 in chondrocytes to inhibit ferroptosisWestern blot and immunofluorescence experiments showed that G1 decreased the phosphorylation level of YAP1 while increasing the expression level of activated YAP1protein and the nuclear translocation of YAP1,thereby increasing the protein expression of FTH1.The YAP1 inhibitor verteporfin inhibited the expression of FTH1 at the transcriptional and protein levels and reduced the inhibitory effect of G1 on ferroptosis in chondrocytes.The YAP1 inhibitor verteporfin inhibited FTH1 expression at both the transcriptional and protein levels and reduced the inhibitory effect of G1 on chondrocyte ferroptosis.It was also found that knockdown of YAP1 expression in chondrocytes significantly attenuated the inhibitory effect of G1 on ferroptosis.8.GPR30 agonist attenuates post-DMM joint damage by inhibiting chondrocyte ferroptosis in miceTransmission electron microscopy analyses of cartilage from OA mouse models showed that mitochondria of osteoarthritic chondrocytes underwent morphological changes characteristic of ferroptosis and that G1 joint injection treatment prevented these ultrastructural and morphological changes.Immunohistochemical staining showed that G1treatment significantly reduced 4-HNE expression and restored FTH1 expression in chondrocytes after DMM in mice.9.GPR30 expression was decreased in osteoarthritic synovial tissues,and GPR30 deficiency exacerbated OA synovial inflammationThe expression of GPR30 in synovial tissues from osteoarthritis patients and DMM model mice was significantly lower than that in normal synovial tissues by immunohistochemical staining and Western blot analysis.The synovitis score and the expression of the synovial matrix degrading enzyme MMP-13 in GPR30 knockout DMM model mice were found to be significantly higher than in wild-type DMM model mice by HE and immunohistochemical staining.10.Agitation of the GPR30 receptor attenuates cartilage degeneration and inhibits synovial inflammation after DMM in miceDMM mouse models of OA were established in wild-type mice and treated with intra-articular injection of G1.G1 not only reduced cartilage degradation,but also lowered synovial inflammation scores in OA mice as assessed by saffron green and HE staining,and reduced MMP-13 expression in synovial tissue as assessed by immunohistochemistry staining.11.G1 decreases expression of cartilage matrix degrading enzymes and inflammatory factors in synoviocytesHuman primary synovial fibroblasts were cultured in vitro and Western blot experiments showed that IL-1βincreased the expression of Adamts5 and MMP-13 in synovial cells in a concentration-dependent manner,whereas G1 was able to reduce the expression level of matrix-degrading enzymes in synoviocytes by inhibiting the NF-κB signaling pathway in them.It was further confirmed by ELISA and q RT-PCR that G1inhibited IL-1β-induced transcript levels and secretion of cartilage matrix degrading enzymes and inflammatory factors in synovial fibroblasts.12.Activation of the GPR30 receptor maintains cartilage extracellular matrix homeostasis by inhibiting the expression of cartilage matrix degrading enzymes and inflammatory factors in fibroblast synoviocytesCo-culture of human fibroblast synoviocytes with primary human chondrocytes,q RT-PCR and Western blot showed that co-culture of chondrocytes with IL-1β-stimulated synoviocytes resulted in a significant increase in the expression of chondrocyte matrix degrading enzymes Adamts5 and MMP-13 and a decrease in the synthesis of chondrocyte matrices COL2 and ACAN,which was reversed by administration of G1.ConclusionIn this study,we found that GPR30 expression was significantly decreased in osteoarthritic articular cartilage and synovial tissues,and GPR30 deficiency exacerbated cartilage degeneration and synovial inflammation after DMM in mice,and agonism of the GPR30 receptor could inhibit synovial inflammation while decreasing cartilage degeneration after DMM in mice.We also found that ferroptosis plays an important role in promoting the OA process,and activation of chondrocyte GPR30 inhibited the phosphorylation of YAP1,increased the expression and nuclear translocation of activated YAP1,and thus increased the transcription and expression of FTH1 to inhibit chondrocyte ferroptosis.Activation of GPR30 receptor can also inhibit the expression of cartilage matrix degrading enzymes and inflammatory factors in synoviocytes through the NF-κB signaling pathway,maintaining the stability of chondrocyte anabolic function,thus playing a role in the prevention and treatment of OA.Therefore,this study investigated the preventive role of GPR30 in OA and its mechanism,providing a theoretical basis for its use as a therapeutic target for OA.