Mechanism Of Chronic Cadmium Exposure Regulating Macrophage Mitochondrial Homeostasis Through RIPK3 To Induce Atherosclerosis

The heavy metal cadmium,as a non-essential element of human body,is widely distributed in the natural environment and is not easy to be degraded.It has been demonstrated that cadmium enter the human body through smoking,cadmium-contaminated diet(rice,fertilizer,sewage),environmental occupational exposure and nickel-cadmium battery pollution.In that way,cadmium has been proved to cause damage to human kidneys,bone and other organs.As for the cardiovascular disease,studies have shown that the vascular wall is becoming one of the target organs of cadmium deposition.Meanwhile,a large number of cross-sectional studies have shown that cadmium exposure is correlated with cardiovascular diseases(such as atherosclerosis,peripheral vascular disease,coronary heart disease,etc.).In addition,recent population studies have confirmed a positive association between blood or urine cadmium concentrations and the development of atherosclerotic plaques in human.However,the underlying mechanism of cadmium-induced atherosclerosis remains unclear.Macrophages,the largest number of immunal cells in the cardiovascular system,are characterized by functional diversity and plasticity.Generally,they have been identified to polarize into Ml or M2 type under different stimuli and play a positive or negative role respectively in the formation of atherosclerotic plaques.In addition,there have been increasing evidences that imbalance of mitochondrial homeostasis is the initial factor of macrophage dysfunction,which is manifested as damage to protective autophagy,promoting the activation of inflammation afterwards.Therefore,this study chose macrophage functional disturbance as the entry point to study the molecular mechanism of atherosclerosis induced by chronic low-level cadmium exposure through macrophage polarization mediated by mitochondrial homeostasis imbalance.In vitro experiments,cadmium chloride was first dissolved in cell culture medium,and cadmium exposure intervention was performed on macrophage RAW264.7 in vitro.After 24h culture,the optimal cadmium exposure concentration was determined as 1μmol/L by cell counting kit(CCK-8)and real-time quantitative polymerase chain reaction(qRT-PCR).Subsequently,we prepared single-cell suspension and extracted proteins from the cells.Flow cytometry demonstrated that cadmium exposure promoted M1-type polarization of macrophages,and western blot biological assay demonstrated that cadmium exposure up-regulated the expression of macrophage nuclear factor kappa B(NF-κB)and NLR family Pyrin domain containing 3(NLRP3)proteins.Finally,proteins in mitochondria were extracted for western blot which demonstrated the results of the up-regulated expression of mitochondrial fission protein Drp1 and down-regulated expression of mitochondrial fusion protein Opal.What’s more,the immunofluorescence staining for mitochondria showed that cadmium exposure resulted in the decreased mitochondrial membrane potential,increased mitochondrial superoxides(mROS)and impaired autophagy,while restoring the mitochondrial homeostasis impaired by cadmium exposure down-regulated the Ml-type polarizarion of macrophages.In the in vivo experiments,cadmium chloride was dissolved in the drinking water of ApoE-/-mice(100mg/L)，and different groups of mice were fed with normal or high-fat diets respectively for 3 months.Subsequently,aortic arches of ApoE-/-mice were taken out and oil red O staining for aortic arch was used to demonstrate the atherosclerotic plaque formation,which was promoted by cadmium exposure with high-fat diet.In addition,we observed the cadmium-induced M1-type polarization and the imbalance of mitochondrial homeostasis in the macrophages of mouse aortic roots by western blot and immunofluorescence staining.Finally,we explored the deep molecular mechanisms of the cadmium-impaired mitochondria and found that receptor interacting serine/threonine kinase 3(RIPK3)and phosphorylated mixed-lineage kinase domain-like protein(p-MLKL)were up-regulated by cadmium exposure.Morever,RIPK3 gene knockout and p-MLKL expression reduction were detected to improve the imbalance of mitochondrial homeostasis,thus effectively reversing the polarization of macrophages and the proliferation of atherosclerotic plaques.In addition,N-acetyl-L-cysteine(NAC)or mitochondrial fission inhibitor 1(Mdivi-1)was used to effectively reduce mitochondrial mROS or mitochondrial fission caused by cadmium,and inhibit the expression of RIPK3 and p-MLKL,finally improving the atherosclerosis and M1-type polarization of macrophages induced by cadmium exposure.In conclusion,we innovatively proposed that chronic low-level cadmium exposure damages mitochondrial homeostasis through the activation of RIPK3 pathway,ultimately leading to Ml-type polarization of macrophages and atherosclerosis.The clear molecular mechanism provides a strong evidence and potential therapeutic targets for cadmium-induced atherosclerosis.