| Dimethoate(DIM)as a widely used medium and low toxic organophosphorus insecticide,can enter the body through the digestive tract,respiratory tract,skin and other ways,and reach all tissues and organs of the body with the blood circulation,causing serious damage to the body.Data shows that about 3 million people around the world have been exposed to organophosphorus environment for a long time,and machine phosphorus pesticides(Organophosphorus pesticides,OPs)directly or indirectly lead to the poisoning or death of about 300,000 people every year.Today,organophosphorus pesticide(Organophosphorus pesticides,OPs)pollution and poisoning is still a global environmental and public health problem.A number of studies have confirmed the damage caused by acute exposure to OPs and paid little attention to the effects of long-term exposure at low dose.In a previous study,we found that DIM exposure by skin can remain in mouse skeletal muscle,leading to oxidative stress in mouse skeletal muscle,and stimulate the apoptotic pathway,inducing apoptosis in C2C12 cells,and NAC can relieve oxidative stress and cell apoptosis caused by DIM.But the effect of DIM exposure on myogenic differentiation of skeletal muscle has not been revealed.Numerous studies and clinical data have confirmed that catechins can increase skeletal muscle mass and strength and promote the differentiation of myogenic stem cells,but whether it is protective during skeletal muscle injury due to DIM is unknown.In this experiment,ICR mice and C2C12 cells were used to further reveal the muscular toxicity of DIM from the perspective of ER stress and to explore the role of EGCG(epigallocatechin-3-gallate)in this process.1.Effect of dimethoate and EGCG on ER stress and myogenic differentiation in mouse skeletal muscleTo explore the effects of DIM and EGCG on ER stress and myogenic differentiation in mouse skeletal muscle.32 male ICR mice were randomized into CON(0.2mL saline intraperitoneal,0.1mL saline on back),EGCG(0.2mLEGCG intraperitoneal,0.1 mL saline on back),DIM(0.2mL saline intraperitoneal,0.1mL DIM on back),DIM+EGCG(0.2mLEGCG intraperitoneal,and 0.1mL DIM on back).Water intake,feed intake,and body weight changes were recorded;the serum CK and LDH activity;Histological changes of skeletal muscle were observed by HE staining;its ultrastructure was observed by transmission electron microscopy;Western blot The expression levels of Bip,PERK/eIF2a pathway-related proteins as well as myogenic differentiation-related proteins MyOD、MyOG、MyHC were determined.Results show that:Compared with the CON group,there was no significant difference in water intake from weeks 1 to 6,and a significant decrease in weeks 7 to 8(P<0.01);overall food intake and body weight were lower in the EGCG and DIM groups than in the CON group;serum CK and LDH activity were significantly increased in DIM group(P<0.01);the skeletal muscle intermuscular space is enlarged and the ultrastructural damage;The phosphorylated protein expression levels of Bip and PERK/eIF2α pathway was significantly increased(P<0.05);the expression level of key proteins for myogenic differentiation was extremely significantly reduced(P<0.01).Compared with the DIM group,no significant changes in water intake and body weight in DIM+EGCG group;significant or very significant decrease in serum CK and LDH activity(P<0.05)or(P<0.01);the intermuscular space in skeletal muscle was narrowed and the ultrastructural damage was somewhat relieved;Bip and PERK/eIF2α pathway phosphoproteins were expressed significantly or significantly reduced(P<0.05)or(P<0.01);the expression levels of key proteins for myogenic differentiation were significantly or highly elevated(P<0.05)or(P<0.01).The results showed that DIM exposure caused mouse skeletal muscle damage,caused ER stress and activated the downstream PERK/eIF2αpathway,and impaired the differentiation of mouse skeletal muscle.EGCG can reduce the activity of serum CK and LDH,significantly inhibit the ER stress and the downstream phosphorylation level of PERK/eIF2α pathway,promote the expression of myogenic differentiation protein,and improve the damage caused by DIM.2.Effects of dimethoate on ER stress and myogenic differentiation in C2C12 cellsTo explore the effect of DIM on ER stress and myogenic differentiation in C2C12 cells.C2C12 cells were treated with different concentrations of DIM(0mM,0.3mM,0.6mM,0.9mM)for 24h.Cell viability was measured by CCK-8,cell morphology and ultrastructure were observed by light and transmission electron microscopy,intracellular Ca2+concentration was measured by Fluo-4 AM Ca2+fluorescent probe,and the expression levels of ER stress marker protein Bip and PERK/eIF2α pathway related proteins in C2C12 cells were determined by Western blot.C2C12 cells were induced to differentiate with culture medium containing 2%horse serum for 48h,and the expression levels of myogenic differentiation-related proteins MyOD,MyOG,and MyHC were measured.The results showed that compared with the CON group,the 0.3mM group had cell viability;intracellular Ca2+ concentration;expression level of Bip and phosphorylation protein of PERK/eIF2αpathway;there was no significant difference in the expression level of myogenic differentiation related proteins.the cell viability of the 0.6mM and 0.9mM groups was significantly reduced(P<0.01);as the concentration of dimethoate increases,cell morphology changes and dead cells increase,resulting in significant damage to cell ultrastructure;the intracellular Ca2+ fluorescence intensity significantly increased(P<0.01);the expression levels of Bip and PERK/eIF2α pathway were significantly or significantly increased(P<0.05)or(P<0.01);the expression level of myogenic differentiation related proteins was significantly or extremely significantly reduced(P<0.05)or(P<0.01).After co-treatment with PERK/eIF2α pathway specific inhibitor GSK2606414,the protein expression levels of DIM+GSK group Bip,p-PERK and p-eIF2α were extremely significantly reduced in the group compared with the DIM group(P<0.01);that of myogenic differentiation was significantly increased compared with DIM group(P<0.01).The results showed that DIM causes ERS in C2C12 cells and activated the downstream PERK/eIF2α pathway and impaired myogenic differentiation the expression levels of relevant proteins were reversed after the addition of the inhibitor,indicating that the PERK/eIF2α pathway mediates the process of impaired myogenic differentiation of C2C12 cells caused by DIM3.Protective effect of EGCG on induced ER stress and impaired myogenic differentiation in C2C12 cellsTo explore the protective effect of EGCG against ER stress and impaired myogenic differentiation in C2C12 cells.In this experiment,CCK-8 was used to screen the concentration and time of EGCG,and then treated with DIM(0.9mM)for 24h.Cell morphology and ultrastructure were observed by light and transmission electron microscopy,intracellular Ca2+concentration was measured by Fluo-4 AM Ca2+fluorescent probe,and the expression levels of Bip and PERK/eIF2α pathway in C2C12 cells were measured by Western blot.Cell differentiation was induced with 2%horse serum for 48h.The expression levels of myogenic differentiation-related proteins MyOD,MyOG and MyHC were detected.Results showed that:compared to the CON group,EGCG inhibits cell proliferation in a concentration-and time-dependent manner,there was no significant difference in cell viability between 10μmol,20μmol,and 30μmol groups for each treatment period,cell viability was significantly reduced in both the 40μmol and 50μmol groups(P<0.01);there were no obvious abnormalities in cell morphology in EGCG group(10 μmol),the ER structure is complete;intracellular Ca2+fluorescence intensity;Bip and PERK/eIF2α pathway related proteins;no significant difference in the expression levels of myogenic differentiation related proteins MyOD,MyOG,and MyHC.In the DIM group,more dead cells,altered cell morphology,and severe destruction of endoplasmic reticulum;intracellular Ca2+fluorescence intensity increased(P<0.01);the expression levels of Bip and PERK/eIF2αpathway related proteins increased(P<0.01);the expression level of myogenic differentiation-related proteins was very significantly reduced(P<0.01).Compared with the DIM group,no dead cells in the co-treated group,but no significant change in cell morphology,and the endoplasmic reticulum structure was complete,but still swelling;the intracellular Ca2+ fluorescence was significantly decreased(P<0.05);the expression levels of Bip and PERK/eIF2α pathway were significantly decreased(P<0.01);the expression level of myogenic differentiation was significantly increased(P<0.05).After co-treatment with PERK/eIF2α pathway specific inhibitor GSK2606414,the expression levels of Bip and PERK/eIF2α pathway-related proteins were significantly reduced in DIM+EGCG+GSK group compared with DIM+EGCG group(P<0.01);the expression levels of myogenic differentiation-related proteins were significantly or very significantly increased(P<0.05)or(P<0.01).The results showed that EGCG promotes myogenic differentiation by inhibiting the expression of PERK/eIF2α pathway-related proteins and thus plays a protective role in the impaired myogenic differentiation of C2C12 cells induced by DIM. |
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