| Dimethoate is one of the most common organophosphorus pesticides now.Once it enters the body,it can cause a variety of tissue and organ’s damage.At present,organophosphorus pesticide poisoning is still a public concern around the world.Because dimethoate is a fat-soluble substance,it can enter the body through undamaged skin and cause damage to the body,but its damage to the body through skin exposure has not been studied.Skeletal muscle is the most abundant tissue in the body,which has the function of supporting body movement,maintaining posture,and adapting to metabolism.As a common sulfhydryl donor,NAC has the effects of anti-oxidation,anti-inflammatory and anti-apoptosis,and has been widely used in clinical practice.This test investigates the effects of dimethoate on the oxidative damage and apoptosis of skeletal muscle cells and the protective effect of N-acetylcysteine in vivo and vitro,further reveal the muscle toxicity of dimethoate and provide a theoretical basis for the safety protection of dimethoate in contact with people and animals.1.Study on the Dimethoate-induced oxidative damage of C2C12 cellsIn order to explore the effect of dimethoate on the oxidative stress and apoptosis of C2C12 cells,C2C12 cells were treated with different concentrations of DIM for 24h and cell viability was detected by CCK-8 method and real-time label-free cell analysis(RTCA);Immunofluorescence method was used to observe the morphology of the nucleus and cytoskeleton;Transmission electron microscope was used to observe cell ultrastructure;The kit was used to detect MDA content and the activity of antioxidant enzymes SOD、GSH-Px、CAT;Westren Blot was used to detect the expression of apoptosis-related protein.The results showed that:compared with the control group,the survival rate of cells treated with 0.6 mM dimethoate for 24h was significantly reduced(P<0.01);The cell nucleus and cell ultrastructure were damaged in a dose-dependent manner;As the concentration of dimethoate increased,the content of MDA increased significantly or extremely significantly(P<0.05 or P<0.01),SOD,GSH-Px,and CAT activity decreased significantly or extremely significantly(P<0.05 or P<0.01);the ratio of Bax/Bcl-2 in the DIM treatment group Significantly or extremely significantly increased(P<0.05 or P<0.01),and the expression of Cleaved-caspase-3 and Cleaved-caspase-9 protein was significantly or extremely significantly increased(P<0.05 or P<0.01).The results showed that dimethoate affects the proliferation of C2C12 cells,damages cell morphology and ultrastructure,induces oxidative stress in C2C12 cells and promotes cell apoptosis.2.The protective effect of N-acetylcysteine in dimethoate-induced oxidative damage of C2C12 cellsIn order to explore the protective effect of N-acetylcysteine in dimethoate-induced oxidative damage of C2C12 cells,this study used 0.6 mM dimethoate and 0.4 mM N-acetylcysteine to treat cells alone or together.cell viability was detected by CCK-8 method and real-time label-free cell analysis(RTCA);Immunofluorescence method was used to observe the morphology of the nucleus and cytoskeleton;Transmission electron microscope was used to observe cell ultrastructure,;The kit was used to detect MDA content and the activity of antioxidant enzymes SOD、GSH-Px、CAT;Westren Blot was used to detect the expression of apoptosis-related protein.The results showed that:compared with the dimethoate group,the cell survival rate of the co-treated group was significantly increased(P<0.05);The damage to the nucleus and mitochondria was relieved;The content of MDA was extremely significantly decreased(P<0.01),SOD,GSH-Px and CAT activity increased significantly(P<0.05);the ratio of Bax/Bcl-2 decreased extremely significantly(P<0.01),and the expression of Cleaved-caspase-3 and Cleaved-caspase-9 protein decreased significantly(P<0.05).The results showed that:NAC can reduce the degree of damage to C2C12 cells caused by dimethoate,restore cell proliferation activity and structure and relieve cell oxidative damage and cell apoptosis caused by dimethoate.3.Study on the oxidative damage of dimethoate-induced skeletal muscle in miceIn vivo experiments were used to further verify the oxidative damage of dimethoate-induced skeletal muscle in mice.32 male Balb/c mice were adapted to rearing for 1 week and then weighed and randomly divided into four groups.The back hair was shaved and 0.1 mL of normal saline or low,medium and high doses of dimethoate solution(20,50,100 mg/kg,respectively)was applied regularly every day,once a day for 15 consecutive weeks.The weight,water consumption and feed intake of the mice were recorded every week.After the test,fasting for 12 hours,the eyeballs were removed and blood was collected to prepare serum;the limbs of the mice were separated by autopsy,fat,fascia and bones were removed,and skeletal muscles were collected.One part is stored in the refrigerator at-80℃,one part is fixed with 4%paraformaldehyde fixative,and the other part is fixed with 2.5%glutaraldehyde fixative.The content of dimethoate in skeletal muscle was detected by gas chromatography,the activity of serum creatine kinase(CK)and lactate dehydrogenase(LDH)was measured by biochemical analyzer,the histological changes of skeletal muscle were observed by paraffin sections,and the ultrastructure changes of skeletal muscle were observed by transmission electron microscope.The kit detects skeletal muscle-related antioxidant indicators,and Westren Blot detects the expression of skeletal muscle apoptosis-related proteins.The results showed that:compared with the control group,there was no significant difference in the weight,diet intake and water consumption of the dimethoate treatment group(P>0.05);there was no dimethoate residue in the skeletal muscle of the control group,while the dimethoate treatment group had no significant difference.The dimethoate content increased in a dose-dependent manner;compared with the control group,with the increase of dimethoate dose,the serum CK activity of mice increased significantly(P<0.05);there was no significant difference in serum LDH activity(P>0.05);the results of paraffin section no obvious lesions were seen;the ultrastructure showed that the nuclei of the dimethoate treatment group shrank,deformed,the mitochondrial cristae was broken,blurred,or even disappeared,and a large number of mitochondria were vacuolated;the content of MDA increased significantly(P<0.01),SOD,GSH-Px,CAT vitality decreased significantly or extremely significantly(P<0.05 or P<0.01);the ratio of Bax/Bcl-2 in the DIM treatment group increased significantly or extremely significantly(P<0.05 or P<0.01),Cleaved-caspase-3 and Cleaved-caspase-9 protein expression increased significantly or extremely significantly(P<0.05 or P<0.01).The results show that dimethoate can remain in the skeletal muscle of mice through skin exposure,and cause skeletal muscle oxidative damage and cell apoptosis.4.The protective effect of N-acetylcysteine in dimethoate-induced oxidative damage of skeletal muscle in miceIn vivo experiments were used to further verify the role of N-acetylcysteine in the oxidative damage of dimethoate-induced skeletal muscle in mice.32 male Balb/c mice were reared for 1 week and their back hair was shaved and randomly divided into four groups:Control group:Apply 100 mL of normal saline on the back once a day,and drink ordinary water freely.DIM group:apply 100mL of 100mg/kg DIM solution on the back once a day,and drink ordinary water freely.DIM+NAC group:apply 100mL of 100mg/kg DIM solution on the back once a day and drink NAC water freely;NAC group:apply 100mL of normal saline on the back once a day and drink NAC water freely for 15 weeks.The weight,water consumption and feed intake of the mice were recorded every week.After the test,fasting for 12 hours,the eyeballs were removed and blood was collected to prepare serum;the mouse limbs were separated by autopsy,fat,fascia and bones were removed,and skeletal muscles were collected.One part is stored in the refrigerator at-80℃,one part is fixed with 4%paraformaldehyde fixative,and the other part is fixed with 2.5%glutaraldehyde fixative.The content of dimethoate in skeletal muscle was detected by gas chromatography,the activity of serum creatine kinase(CK)and lactate dehydrogenase(LDH)was measured by biochemical analyzer,the histological changes of skeletal muscle were observed by paraffin sections,and the ultrastructure changes of skeletal muscle were observed by transmission electron microscope.,The kit detects skeletal muscle-related antioxidant indicators,and Westren Blot detects the expression of skeletal muscle apoptosis-related proteins.The results showed that there were no significant differences in body weight,dietary intake and water consumption between the DIM+NAC group and the DIM group(P>0.05);the content of dimethoate in skeletal muscle was not significantly different(P>0.05);CK activity decreased significantly(P<0.05),serum LDH activity did not change significantly;paraffin section results showed no obvious lesions;ultrastructure showed that nucleus shrinkage was relieved,mitochondrial morphology and structure were basically normal;MDA content was extremely significantly decreased(P<0.01),the vitality of SOD,GSH-Px,and CAT increased significantly or extremely significantly(P<0.05 or P<0.01);the ratio of Bax/Bcl-2 decreased extremely significantly(P<0.01),Cleaved-caspase-3 and Cleaved-caspase-9 protein expression decreased significantly(P<0.05).The results show that:N-acetylcysteine can alleviate the oxidative damage and apoptosis of skeletal muscle caused by dimethoate. |
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