Effects Of Cadmium Chloride On Immunotoxicity And Th1/Th2/Th17/Treg Cell Balance Of Rats

Part ? Immunotoxic Effect of Subchronic Cadmium Exposure on SD RatsObjective:To establish animal exposure models to observe the effects of subchronic cadmium exposure on the indexes of blood routine,functions of livers and kidneys.The histopathological changes of liver,kidney,lung,thymus,spleen and mesenteric lymph nodes were also observed,so as to explore long-term low-dose cadmium exposure on immunotoxic effects.Method:Forty SD rats,half male and half female,with an average body weight of?284.71±55.95?g,were randomly divided into 4 groups:the Control group?ddH2O?,the CdCl₂ 75 mg/L group,150 mg/L group,and 300 mg/L group.Feed the rats with standard fodder regularly,give free access to food and drinking water.The control group drank pure double distilled water.The cadmium-dose group drank CdCl₂ double distilled water.Then they were poisoned for another 9 months.Their weights were measured and recorded every week.At the end of the experiment,the rats'blood was collected from abdominal aorta under anesthesia.And the liver,kidney,lung,thymus,spleen,mesenteric lymph nodes and other organs were taken after blood collection.They were weighed and the organ coefficients were calculated.Some tissues were stained and sectioned.The pathological changes of various organ tissues were observed and analyzed.Indexes of blood routine and liver and kidney function were detected by automatic blood routine detector and automatic biochemical analyzer.Results:The body weight of rats in each dose group increased with time?P<0.05?,but there were no significant differences in body weight between the groups at each time point?P>0.05?.There were no significant differences in liver,kidney,lung and spleen quality and organ coefficients in each dose group?P>0.05?.The thymus mass and organ coefficients were significantly higher in 75 mg/L and 150 mg/L dose groups,and the difference was statistically significant?P<0.05?.);The results of blood routine indicators showed that compared with the control group,the WBC,LYMPH and PLT in 300 mg/L dose group were significantly increased?P<0.05?,and the HGB in 150 mg/L and 300 mg/L dose groups significantly decreased?P<0.05?.Compared with 75mg/L dose group,The WBC and PLT of the 300 mg/L dose group were significantly elevated?P<0.05?;The results of liver and kidney function indicators showed that compared with the control group,the 150 mg/L dose group had higher AST,UREA,UA and the 300 mg/L dose group had significantly increased ALT,AST,UREA,CREA and UA?P<0.05??P<0.05?.Compared with 75mg/L dose group,the 150 mg/L dose group had increased UA significantly and the ALT,AST,UREA,CREA of the 300mg/L dose group increased significantly?P<0.05?.Histopathological examination showed that the liver tissue of each group had different degrees of congestion,edema,with necrosis and regeneration,and the tubular epithelial cells of the renal tissue were swollen with vacuolar degeneration and necrosis.There was pulmonary interstitial fibrosis in the lung tissue with widening in the lung septum,occlusion and disappearance of the interstitial vessels,and diminish in the alveolar space.There were lymphocytic reactive hyperplasia in the thymus and mesenteric lymph nodes,enlarged follicular germinal center,and increased phagocytic cells.Spleen white pulp atrophied and the lymphocytes in the lymphatic sheath and marginal zone around the artery decreased with expansion and congestion of red blood marrow sinus.THe phagocytosis increased and the 300 mg/L dose group had red pulp partial fibrosis.The degree of lymphadenopathy in liver,kidney,lung,thymus,spleen and mesentery gradually increased with the increase of dose.Conclusion:?1?Subchronic cadmium exposure can cause liver,kidney and lung damage to varying degrees.?2?Subchronic cadmium exposure can cause changes in blood routine indicators and damage to thymus,spleen and mesenteric lymph nodes in rats.Part ? Effect of Cadmium exposure on the Balance of Th1/Th2/Th17/TregObjective: More and more evidence shows that cadmium can induce autoimmune diseases,but its mechanism is not clear.The purpose of this study is to detect the expression of major transcription factors of subgroup cells Th1/Th2/Th17/Treg of lymphocytes CD4+T and related cytokines after being exposed to cadmium chloride at cellular and animal levels,and to explore the effects of cadmium on Th1/Th2/Th17/Treg balance and its role in inducing autoimmune diseases.Method: This experiment uses Jurkat T lymphocytes as an in vitro experimental object.The Jurkat T lymphocytes were exposed to different concentrations?0?mol/L,5?mol/L,10?mol/L,20?mol/L,30?mol/L,40?mol/L,50?mol/L?of Cd Cl2 for 24 h,48h and 72 h.Observe the growth pattern and morphological changes of each group of cells.The proliferation inhibition rates of each group of cells were detected by CCK8 kit.E The Jurkat T lymphocytes were exposed to low-dose cadmium with concentrations of 0?mol/L,5?mol/L,10?mol/L and 20?mol/L with half-inhibition rate?IC50?or lower rate.The exposure time included 24 h,48h,72 h.After being poisoned,the rats' RNA were extracted and transcribed and synthesized as c DNA.Real-time PCR technology was adopted to detect the m RNA expression level of genes T-bet?GATA3?RORC?Foxp3?TGF-??IL-6?IL-16?IL-23.?2?The blood sample of the SD rats in Part I was used as an experimental object in vivo.The antibody concentration of Ig G,Ig M and the protein levels of cytokines IL-1a,IL-1?,IL-4,IL-5,IL-6,IL-17 A,IL-17 F and TNFa in plasma were detected by ELISA kit.RNA was extracted from whole blood.Detect the the CD4+T lymphoid Th1,Th2,Th17,Treg of the four subtypes of the main transcription factors T-bet,GATA3,RORc,Foxp3 and the gene mRNA expression change of corresponding cytokines IFN-r?IL-2?IL-4?IL-6?IL-17A?IL-17 F ?IL-22?IL-10 ?TGF-?.Results: 1.Real-time PCR results of Jurkat T lymphocyte:The m RNA expression results of four major subtypes of transcription genes Th1,Th2,Th17 and Treg in Jurkat T lymphocytes showed that:Compared with the control group,T-bet?GATA3 and RORc gene expression did not change significantly?P>0.05?;and the expression of Foxp3 genes exposed to poison for 72 h decreased in each dose group?P<0.05?.The expression results of Cytokine m RNA showed that:Compared with the control group,the expression of TGF-? gene decreased in each dose group?P<0.05?;IL-6 gene expression increased in 20?mol/L dose group in all time periods?P<0.05?;IL-16 gene expression had no significant change?P>0.05?,and the expression of IL-23 gene increased at 72h?P<0.05?.2.?1?The expression of antibodies and cytokines in peripheral blood of SD rats was detected by ELISA: Compared with the control group,the 300mg/L dose group had increased the centration of Ig G and the difference was significant?P<0.05?.The results of cytokine protein expression showed:Compared with the control group,the IL-1a expression of the 300mg/L dose group increased significantly?P<0.05?.The expression of IL-1beta?IL-4?IL-5?IL-17F?TNFa had not changed obviously?P>0.05?;the expression of IL--17 A in the 300mg/L dose group increased?P<0.05?.?2?Real-time PCR was used to detect the gene expression in peripheral blood of SD rats:Compared with the control group,the T-bet gene did not change significantly?P>0.05?,the GATA3 gene in 150mg/L dose group increased?P<0.05?,and the RORc gene in 150mg/L and 300mg/L dose groups increased?P<0.05?.The expression of Foxp3 gene was significantly decreased in each dose group?P<0.05?,and the RORC/Foxp3 ratio was significantly increased in each dose group?P<0.05?.The expressions of IFN-r,IL-2 and IL-22 genes were all expressed in each dose group without significant change?P>0.05?.The expression of IL-4 gene in 300mg/L dose group decreased?P<0.05?,and the 300mg/L doses of IL-6,IL-17 A and IL-17 F genes were significantly increased?P<0.05?.The expression of IL-10 gene was significantly decreased in each dose group?P<0.05?.The expression of TGF-beta gene in 150mg/L and 300mg/L dose groups decreased significantly?P<0.05??Conclusion:?1?Cadmium exposure can cause imbalance of Th1/Th2/Th17/Treg cells in Jurkat T lymphocytes and peripheral blood of rats,mainly upregulation of Th17/Treg cells.?2?Th17/Treg cell imbalance caused by cadmium exposure may increase the risk of cadmium-induced autoimmune diseases.