Mechanism Of CaMKⅡ/PGC-1α Pathway In Dimethoate-Induced Insulin Resistance Of Chicken Skeletal Muscle Satellite Cells

As a large agricultural country,China uses a lot of pesticides.Since organochlorine pesticides were banned,organophosphorus pesticides have become the mainstream of the market,and with the continuous development of the economy,the application of pesticides is not only limited to agriculture,but also widely used in insecticide and sterilization in daily life.Dimethoate having as a low cost,broad insecticidal spectrum,low toxicity of organophosphorus pesticides is widely used in agriculture and non-agriculture.Acute toxicity on organophosphate pesticides have been through numerous studies and clinical cases illustrate,but long-term low-dose how the body is the presence of toxic effects is still controversial.It has been found organophosphorus pesticide poisoning can cause symptoms such as high blood sugar and urine sugar,but the exact pathogenesis is not yet clear;However,glucose and lipid metabolism disorders are closely related to mitochondrial dysfunction,but whether there are linkages between the three is not yet known.Therefore,this study took primary chicken skeletal muscle satellite cells as the research object,and explored the mechanism of CaMKII/PGC-1α pathway in chicken skeletal muscle satellite cells’ antioxidant capacity,mitochondrial biosynthesis and maintenance of glucose metabolism balance,provide a certain theoretical basis for dimethoate-induced insulin resistance in chicken skeletal muscle satellite cells.1.Optimization and identification of the isolation and purification method of primary chicken skeletal muscle satellite cells.The purpose of the study was to explore the techniques for isolation,culture,purification,differentiation and identification of chicken skeletal muscle satellite cells(MSC)in vitro;in this experiment,we selected SPF chicken embryos at 12 embryonic ages,integrated loose muscle fibers and digested free cells and other principles,used self-made mixed enzyme digestion method to separate cells,and performed a series of optimizations for subsequent cell purification,in vitro culture,and differentiation;Simultaneous detection of morphological identification,labeling and immune-specific genes resulting in three aspects Purification cells were identified,in order to establish self-separation mixing one kind of enzyme primary chicken skeletal muscle satellite cells.The results show that the separated and purified cells have strong refractive index,adhere to the wall after 24 hours and are spindle-shaped;after induced differentiation,the cells fuse and form neatly arranged myotubes;the cell survival rate after the optimized separation method was(90.82±1.294)%,and the cell purity after the optimized purification method was(90.44± 1.264)%;immunolabeling assay marker protein Pax7,Desmin positive;qRT-PCR detection of the marker genes Pax7,MyHC,MyoD1 were positive,and the transcription level of Pax7 gene in quiescent phase was 1.705 times that after differentiation,while the transcription level of MyHC gene in differentiation phase was 13.073 times that of quiescent phase;The results showd that this experiment established a fast and convenient self-made mixed enzyme digestion method,which laid the foundation for basic research on chicken skeletal muscle,meat product improvement and subsequent experiments.2.Study on toxic injury of primary chicken skeletal muscle satellite cells induced by dimethoate.To explore the toxic effect of dimethoate on primary chicken skeletal muscle satellite cells.in this experiment,primary chicken skeletal muscle satellite cells were used as the object,and different concentrations of dimethoate(0,0.125,0.25,0.5,1mM)were used for 12h and 24h treatments respectively.The CCK-8 was used to detect the effect of dimethoate on the proliferation of primary chicken skeletal muscle satellite cells;observe the influence of dimethoate on cell morphology with light microscope and scanning electron microscope;microplate method to detect MDA content and T-SOD,CK enzyme activity;flow cytometry to detect ROS content;chemiluminescence method to detect the level of Ca2+ inside and outside the cytoplasm;qRT-PCR detects the transcription levels of MyHC,MSTN and HSP70.The results show that compared with the control group,0.125mM dimethoate has no effect on cell proliferation,while 0.25mM dimethoate has a significant inhibitory effect on cell growth(P<0.05);With the increase of dimethoate concentration,the phenomenon of myotubes breaking,stringing and widening can be observed;after the cells were treated with 0.25,0.5mM dimethoate for 12h and 24h,the MDA content and CK enzyme activity all increased significantly(P<0.01);CAT and T-SOD enzyme activities increased extremely significantly after 0.25mM dimethoate treatment(P<0.01);after treatment with 0.5mM dimethoate,it was extremely significantly decreased(P<0.01);the content of ROS and Ca2+ in the cytoplasm increased significantly(P<0.01);the transcription levels of MyHC and MSTN genes showed significant(P<0.05)and extremely significant declines(P<0.01)after treatment with 0.25mM and 0.5mM dimethoate;the transcription level of Hsp70 gene decreased significantly(P<0.05)after 0.25mM,0.5mM dimethoate treatment for 12h,and after 24h it showed an extremely significant increase(P<0.01).The results showd that dimethoate can cause proliferation and morphological damage of primary chicken skeletal muscle satellite cells,and induce muscle damage and oxidative stress.3.Study on mitochondrial damage of primary chicken skeletal muscle satellite cells induced by dimethoate.To investigate the mechanism of dimethoate-induced mitochondrial damage in primary chicken skeletal muscle satellite cells,the cells were treated different concentration of DIM(0,0.25,0.5mM)for 12h and 24h.Detection of CS enzyme activity by microplate method;flow cytometry to detect Ca2+level in mitochondria;western blot detection of Cyt-c protein expression level;mito-Tracker Green probe is loaded to observe mitochondrial morphology and transmission electron microscope to observe mitochondrial ultrastructure;qRT-PCR detection of mitochondrial biosynthesis and fusion division related gene transcription level.The results show that compared with the control group,after 0.25,0.5mM dimethoate treated cells for 12h and 24h,the CS enzyme activity showed a downward trend at 12h after dimethoate treatment,and showed a significant increase at 24h(P<0.05);the level of Ca2+ in mitochondria showed an extremely significant increase(P<0.01);the expression level of Cyt-c protein decreased significantly when treated with 0.5mM dimethoate for 12h(P<0.05),while it showed an extremely significant increase when treated with 24h(P<0.01);the morphology of mitochondria appeared fragmentation and dispersion,transmission electron microscopy showed that the internal mitochondria were vacuolated and the mitochondrial cristae were blurred;the transcription levels of mitochondrial biosynthesis-related genes Tfam and Nrf-1 increased significantly(P<0.01)after 12h treatment with 0.5mM DIM,and decreased extremely significantly after 24h treatment(P<0.01);the transcription levels of mitochondrial division and fusion related genes Mfn-1,Mfn-2,Opa-1,and Drp-1 showed an extremely significant increase(P<0.01).The results showd that DIM caused significant changes in the internal and external morphology of primary chicken skeletal muscle satellite cells,and inhibited the transcription level of mitochondrial biosynthesis-related genes.At the same time,it induced mitochondrial fusion and division disorder,which eventually led to mitochondrial damage in primary chicken skeletal muscle satellite cells.4.Study on the mechanism of DIM induced insulin resistance in primary chicken skeletal muscle satellite cellsTo investigate the mechanism of DIM induced insulin resistance in primary chicken skeletal muscle satellite cells,the cells were treated different concentration of DIM(0,0.25,0.5mM)for 12h and 24h.Using a microplate assay ATP and protein carbonyl content;qRT-PCR detects the transcription levels of calcium channel-related genes CaMKII,PGC-1α,PKA,PP2A;flow cytometry to detect glucose intake level;western blot detection of insulin resistance associated protein IRS-1,AKT,P-AKT expression levels;qRT-PCR detects the transcription level of glucose transporter GLUT1/8/12.The results show that compared with the control group,after 0.25 mM,0.5 mM dimethoate treated cells for 12 and 24 hours,the protein carbonyl and ATP content were extremely significantly increased(P<0.01),and showed a dose-dependent effect;after DIM treatment for 12 hours,the CaMKII transcription level appeared significantly(P<0.05)or extremely significantly decreased(P<0.01),while PGC-1α and PKA showed an extremely significant upward trend(P<0.01),and the PP2A gene showed a significant(P<0.05)or extremely significant increase(P<0.01);after 24h of DIM treatment,the transcription levels of CaMKII and PP2A showed a significant upward trend(P<0.05),and PKA and PGC-1α showed a very significant downward trend(P<0.01);the glucose intake level increased significantly after 0.5mM DIM treatment for 12h(P<0.05),and it decreased extremely significantly at 24h(P<0.01);the expression level of IRS-1 protein increased significantly at 12h(P<0.01),and decreased extremely significantly at 24h(P<0.01);the expression level of P-AKT protein showed an extremely significant decrease(P<0.01);glucose transporter GLUT1/8/12 treatment significantly increase(P<0.01)after 12h at DIM,extremely decreased significantly after 24h(P<0.01).The results showd that low-dose DIM can increase the protein carbonylation level of primary chicken MSC and destroy the Ca2+ homeostasis in mitochondria,by activating the transcription level of CaMKII,inhibit the transcription of downstream mitochondrial synthesis-related gene PGC-1α,down-regulate PKA/IRS-1 expression,at the same time,PP2A inhibits the phosphorylation of AKT so that the insulin signal cannot be transmitted to the glucose transporter,which ultimately leads to insulin resistance in the primary chicken MSC.