Effects Of CRH, ACTH, GC On Immune Function Of Rat Splenic Lymphocytes Exposed To AlCl₃in Vitro

The purpose of this research was to investigate the effects of corticotropin releasing hormone（CRH）, adrenocorticotropic hormone （ACTH） and glutcocorticoid （GC） on immune function of ratlymphocytes exposed to AlCl₃in vitro. The lymphocyte cultured in RPMI-1640medium wereisolated from the spleen of Wistar rats （200～210g） under sterile conditions.There were fivegroups for each hormone.The groups included:CRH Test:0mmol/L AlCl₃+0mol/L CRH （Blank group, C-B）,1/10IC₅0AlCl₃+0mol/L CRH（Control group, C-C）,1/10IC₅0AlCl₃+1×10-10mol/L CRH （Low-dose group, C-L）,1/10IC₅0AlCl₃+1×10^-9mol/L CRH （Middle-dose group, C-M）,1/10IC-850AlCl₃+1×10mol/L CRH（High-dose group, C-H）.ACTH Test:0mmol/L AlCl₃+0mol/L ACTH （Blank group, A-B）,1/10IC₅0AlCl₃+0mol/LACTH （Control group, A-C）,1/10IC₅0AlCl₃+1×10^-13mol/L ACTH （Low-dose group, A-L）,1/10IC₅0AlCl₃+1×10-12mol/L ACTH （Middle-dose group, A-M）,1/10IC₅0AlCl₃+1×10-11mol/LACTH （High-dose group, A-H）.GC Test:0mmol/L AlCl₃+0mol/L GC （Blank group, G-B）,1/10IC₅0AlCl₃+0mol/L GC（Control group, G-C）,1/10IC₅0AlCl₃+1×10^-8mol/L GC （Low-dose group, G-L）,1/10IC₅0AlCl₃+1×10-7mol/L GC （Middle-dose group, G-M）,1/10IC₅0AlCl₃+1×10-6mol/L GC（High-dose group, G-H）.The CCK-8assay was used to detect the IC₅0of aluminium on rat splenic lymphocyte and theproliferation rates of T, B lymphocyte. The T lymphocyte subsets were detected by flow cytometry,secretion of IL-2and TNF-α were detected by radioimmunoassay, secretion of IgG and contents ofcAMP were detected by ELISA and corticotropin releasing hormone receptor1（CRHR1）,adrenocorticotropic hormone receptor （ACTHR） and glutcocorticoid receptor （GCR） mRNAexpression were detected by real-time quantitative reverse transcription PCR （qRT-PCR）, GCRprotein expression was detected by Western Blot. The results were shown as below:1. CRH intervention test:1). The proliferation rates of T, B lymphocyte, the CD3⁺, CD4⁺T lymphocyte express ratesand CD4⁺/CD8⁺value, secretion of IL-2, TNF-α and IgG in C-L group were all higher than in C-Cgroup （P＜0.05; P＜0.01）. The proliferation rates of T, B lymphocyte, the CD3⁺, CD4⁺Tlymphocyte express rates and CD4⁺/CD8⁺value, secretion of IL-2, TNF-α and IgG in C-M groupdidn’t have significant difference compared with C-C group （P＞0.05）. The proliferation rates of T, B lymphocyte, the CD3⁺, CD4⁺T lymphocyte express rates and CD4⁺/CD8⁺value, secretion ofIL-2, TNF-α and IgG in C-H group were all lower than in C-C group （P＜0.05; P＜0.01）. Theresults indicate: Low concentration CRH alleviates immunosuppression of AlCl₃on ratlymphocyte in vitro; High concentration CRH aggravates immunosuppressive of AlCl₃on ratlymphocyte in vitro.2). The contents of cAMP in lymphocyte in C-L group were lower than C-C group （P＜0.05）,the CRHR1mRNA expression in C-L group didn’t have significant difference compared with C-Cgroup（P＞0.05）. The contents of cAMP in lymphocyte in C-M group didn’t have significantdifference compared with C-C group（P＞0.05）, the CRHR1mRNA expression in C-M group werehigher than C-C group （P＜0.01）. Both the contents of cAMP and the CRHR1mRNA expressionin C-H group were higher than C-C group （P＜0.05; P＜0.01）. The results indicate: The“cAMP-CRHR” signal regulatory mechanism of CRH on lymphocyte immune function is disorderin vitro.2. ACTH intervention test:1). The proliferation rates of T, B lymphocyte, the CD3⁺, CD4⁺T lymphocyte express ratesand CD4⁺/CD8⁺value, secretion of IL-2, TNF-α and IgG in A-L group and A-M group didn’t havesignificant difference compared with A-C group （P＞0.05）. The proliferation rates of T, Blymphocyte, the CD3⁺, CD4⁺T lymphocyte express rates and CD4⁺/CD8⁺value, secretion of IL-2,TNF-α and IgG in A-H group were all lower than in A-C group （P＜0.05; P＜0.01）. The resultsindicate: High concentration ACTH aggravates immunosuppressive of AlCl₃on rat lymphocyte invitro.2). The contents of cAMP in lymphocyte in A-L group and A-M group didn’t have significantdifference compared with A-C group （P＞0.05）, the contents of cAMP in A-H group were higherthan A-C group （P＜0.01）. The ACTHR mRNA expression in all groups were higher than A-Cgroup （P＜0.01）. The results indicate: The “cAMP-ACTHR” signal regulatory mechanism ofACTH on lymphocyte immune function is disorder in vitro.3. GC intervention test:1). The proliferation rates of T, B lymphocyte, the CD3⁺, CD4⁺T lymphocyte express ratesand CD4⁺/CD8⁺value, secretion of IL-2, TNF-α and IgG in G-L group were all higher than in G-Cgroup （P＜0.05; P＜0.01）. The proliferation rates of T, B lymphocyte, the CD3⁺, CD4⁺Tlymphocyte express rates and CD4⁺/CD8⁺value, secretion of IL-2, TNF-α and IgG in G-M groupdidn’t have significant difference compared with G-C group （P＞0.05）. The proliferation rates of T,B lymphocyte, the CD3⁺, CD4⁺T lymphocyte express rates and CD4⁺/CD8⁺value, secretion ofIL-2, TNF-α and IgG in G-H group were all lower than in G-C group （P＜0.05; P＜0.01）. Theresults indicate: Low concentration GC alleviates immunosuppression of AlCl₃on rat lymphocytein vitro; High concentration GC aggravates immunosuppressive of AlCl₃on rat lymphocyte invitro.2). The contents of cAMP in lymphocyte in G-L group were lower than G-C group （P＜0.05）, the GCR mRNA and protein expression in G-L group were higher than G-C group （P＜0.05）. Thecontents of cAMP in lymphocyte, the GCR mRNA and protein expression in G-M group didn’thave significant difference compared with G-C group （P＞0.05）. The contents of cAMP inlymphocyte in G-H group were higher than G-C group （P＜0.01）, the GCR mRNA and proteinexpression in G-H group were lower than G-C group （P＜0.05）. The results indicate: Lymphocytehave a kind of immune protect mechanism for itselves by adjusting GCR mRNA and proteinexpression within a certain range.