The Role Of Autophagy In Cadmium-induced Osteocyte Toxicity In Mice

Cadmium(Cd)is a toxic heavy metal and also a pollutant widely present in the environment.Studies have found that the level of cadmium exposure in the environment is closely related to changes in bone mass.Previous studies believed that the balance of bone formation and bone resorption involved in osteoclasts and osteoblasts determines the level of bone mass,and more and more studies have shown that bone cells also play a role in bone anabolism due to their powerful physiological regulation capabilities.Important functions,their activity or quantity changes can cause bone remodeling imbalance and cause osteoporosis diseases.In recent years,studies have found that autophagy plays an important role in the dynamic balance of bone tissue.Autophagy can be used as a protective mechanism to play an adaptive role in protecting the body from various stimuli.Autophagy plays a protective role in diseases such as body ischemia,desmin-related cardiomyopathy,and heart failure caused by atrophy.However,the effect of long-term cadmium exposure on bone damage and the protective effect of autophagy in the process are still unclear.In this experiment,the mouse MLO-Y4 bone cell line was used as a model to study the effect of cadmium on bone damage and the protective effect of autophagy during the process,providing a new idea for preventing and treating the toxic effect of cadmium on bone.1.The effect of cadmium exposure on bone tissue,serum and urine of miceThirty-three 5-week-old female BALB/c mice were randomly divided into three groups and drank cadmium water with a concentration of 0,5,and 25 mg/L respectively for 16 months.The water and food intake were counted every week.After the test,mice serum and urine were collected,and bone tissues were collected by dissection.Determinate serum alkaline phosphatase(ALP)activity,serum calcium and phosphate(Ca,P)and urine cadmium and Ca and P content using biochemical analyzer,atomic absorption spectrophotometric determination of cadmium content in urine,paraffin section observe the pathological changes of hind limb bones,observe the distribution and expression of LC3,ATG5,and ATG7 proteins in hind limb bones by immunohistochemistry.Use metabonomics method to detect the changes of metabolites in mouse serum and urine.Use Western blot method to detect the changes in the expression of autophagy characteristic proteins and PI3K/AKT/mTOR signaling pathway proteins in bone tissue.The results showed that:① As the concentration of cadmium in drinking water increased,there was no significant difference in body weight and dietary intake,while the cadmium intake increased significantly.The content of the ALP activity and urine cadmium,Ca,P in the cadmium-exposed group increased in a dose-dependent manner.Among them,the urine cadmium,P,serum ALP activity in the 5 mg/L group and the 25 mg/L group and the urine Ca content in the 25 mg/L group were significantly different(P<0.05)or extremely Significantly(P<0.01).The increase in serum ALP indicates the increase in bone damage,and the increase in the content of Ca and P in urine indicates that the body’s ability to absorb Ca and P may be weakened;②As the concentration of cadmium in drinking water increases,the bone trabecula becomes thinner and sparse,which indicates that the microstructure of the bone is changed;③ Compared with the control group,the LC3 and ATG5 protein content in the hard bone of the cadmium-exposed group increased,and the ATG7 protein content did not change much,indicating that the bone hard was free The expression of phagocytic characteristic protein changes;④Compared with the control group,the metabolites of the 5 mg/L and 25 mg/L cadmium-exposed groups have a tendency to separate,indicating that there is a difference between the cadmium-exposed group and the control group,of which the serum metabolite is 5 mg/L.There is a crossover between the 25mg/L cadmium-exposed group,and the separation is not significant.The urine metabolite 25mg/L cadmium-exposed group is farther in space than the 5mg/L cadmium-exposed group,and the difference is more significant,indicating that the concentration gradient of cadmium water feeding is small rats have different effects on the metabolites of mice.The more significant in separation,the greater in impact;⑤The steroid hormones and lysine metabolism pathways of the cadmium-exposed group are affected,indicating that cadmium is related to the occurrence of bone metabolism diseases;⑥The expression of autophagy-specific proteins P62,p-PI3K,p-AKT,and p-mTOR in the bone tissue of the 25 mg/L cadmium group were extremely significantly decreased(P<0.01),while the expression of LC3 II was extremely significantly increased(P<0.01),and in the 5 mg/L cadmium group,the expression of P62 protein decreased significantly(P<0.05),and the expression of LC3II protein increased significantly(P<0.01).Conclusion:①Long-term cadmium exposure can reduce the accumulation of Ca and P elements in the animal body,increase the activity of ALP in the serum,destroy the bone microstructure,and cause bone damage;②After prolonged cadmium exposure,the expression of autophagy protein in the bone rigidity increases.High;③Cadmium may aggravate the occurrence of bone metabolism diseases by affecting the metabolites in blood and urine;④The expression of autophagy characteristic proteins in bone tissue increased after cadmium exposure,PI3K/AKT/mTOR signaling pathway indirectly regulates bone tissue cell autophagy.2.The effect of cadmium exposure on mouse MLO-Y4 bone cell lineMLO-Y4 cells were treated with 0,20,40,and 80 μM cadmium concentrations,and the morphological changes of the cells were observed under a microscope,and the expression of autophagy characteristic proteins LC3 and P62 were detected by Western blot.On this basis,Western blot was used to detect the expression of ATG5,ATG7 and PI3K/AKT/mTOR signaling pathway proteins.The autophagy inducer rapamycin was used to observe the changes of cell morphology under a microscope,and the expression of autophagy characteristic proteins LC3 and P62 protein were detected by Western blot..The results showed that compared with the control group,①80uM cadmium treatment showed obvious changes in cell morphology for 6 hours,the cell tree mutation was short,and the cells became round;②The expression of LC3 protein in the 80 μM cadmium treatment group was extremely significantly increased(P<0.01),and the expression of P62 protein was extremely significantly reduced(P<0.01);③The protein expression of ATG7 and ATG5 in the 80uM cadmium treatment group was significantly increased(P<0.05)or extremely significantly(P<0.01);④The expression of p-AKT,p-PI3K and p-mTOR protein in the 80uM cadmium treatment group was significantly decreased(P<0.05)or extremely significantly reduced(P<0.01);⑤Compared with the cadmium-exposed group,the trend of cell morphology changes in the rapamycin and cadmium co-treatment group was relieved;⑥Compared with the control group,the LC3 protein expression in the rapamycin and cadmium co-treatment group was extremely increased(P<0.01)and higher than the cadmium-exposed group,the expression of P62 protein was significantly reduced(P<0.05)and lower than the cadmium-exposed group.Summary:①Prolonged exposure to cadmium can change the morphology of bone cells and cause autophagy.The PI3K/AKT/mTOR signaling pathway can regulate the level of autophagy during the toxicity of cadmium to bone cells.② Autophagy has a protective effect in the process of cadmium toxicity to bone cells,and further enhancing the level of autophagy can alleviate the toxicity of cadmium to bone cells.