Effect Mechanism Of NaF On Splenic Cell Proliferation,Apoptosis,Autophagy And Immunity In Mouse

Fluorine is one of the obligatory trace elements for organism,which is extensive exist in water,air,soil,rocks and other ecological environments.Appropriate amount of fluorine intake has a positive effect on the prevention of dental caries and bone formation,but long-term excessive intake of fluorine may cause severe skeletal and non-skeletal fluorosis.Spleen is a peripheral immune organ of human and animal,it is the main place of immune responses and is one of the target organs of fluorosis.Studies have revealed that excessive fluorine can induce a series of damage such as oxidative stress,cell cycle arrest,apoptosis,and immunosuppression,but there is still a lack of systematic research report.Sodium fluoride(NaF)is a common compound and used widely in nature,which is easily affects humans and animals.Up to now,there is no systematic research on the mechanism of NaF-damaged splenic function in animals and human.So this study was conducted to clarify the potential molecular mechanism of NaF-damaged spleen through a fluorosis model in vivo,which was on the basis of cell culture in vitro.Aim to observe the changes of histological structure,cell cycle,T and B cell subset,immune response,ER stress,apoptosis and autophagy using experimental pathology,immunohistochemistry,flow cytometry,q RT-PCR,ELISA,and western blot,which provided new data reference and theoretical basis for further study on the toxicity effects of NaF on spleen.The research contents and results are as follows:EXP.1 Effects of NaF on splenic lymphocyte proliferation in mice in vitroSplenic lymphocytes were isolated from 4-week-old male ICR mice and exposed to sodium fluoride(0,100,500,and 1000?mol/L)for 24h.The splenic lymphocyte proliferation was examined after 24 h of the experiment.The results showed that NaF in excess of 500?mol/L increased the cytotoxicity(LDH release,cell mortality),decreased T and B cell viability(OD value)and the proportion of CD3⁺,CD3⁺CD4⁺,CD3⁺CD8⁺and CD19⁺,which further reduced the expression levels of related cytokines(IL-2,TGF-?,TNF-?,IFN-?)and increased IL-10 levels by a dose-dependent manner.The FACs results showed that NaF in excess of 500?mol/L arrested G0/G1 cell cycle,which characterized by increasing cell percentage at G0/G1 phase,reducing the cell percentages of S and G2/M phase.Concurrently,protein expression levels of CDK4,Cyclin D and CDK2,Cyclin E were significantly decreased,but CDK1,cyclin A and cyclin B were not changed,implying that the cell cycle was blocked in G0/G1 phase.EXP.2 Effects of NaF on splenic cell proliferation in mice in vivo1.Effects of NaF on splenic histological structure240 ICR mice were equally allocated into four groups with intragastric administration of NaF solution:0 mg/kg,12 mg/kg,24 mg/kg and 48 mg/kg for 42 days.The pathological observation was conducted at the 21^st and 42^nd day of the experiment.The results showed that NaF in excess of 12 mg/kg resulted in histopathological lesions,which specifically expressed as decreasing the splenic volume and growth index.The lymphocytes in the white and red pulp were significantly reduced with a dose-and time-dependent manner in the three NaF-treated groups during 42-day experiment.2.Effects of NaF on splenic cell cycleAt the 21^st and 42^nd day of the experiment,FACs results showed that NaF in excess of12 mg/kg increased the cell percentage at G0/G1 phase,decreased cell percentages at S and G2/M phase in the NaF-treated groups,which indicated that cell cycle was arrested in G0/G1 phase.The q RT-PCR and western blot methods were used to determine the key regulatory molecules that mediated cell cycle from G1 to S phase.It was found that the m RNA and protein expression levels of CDK2,Cyclin E and CDK4,Cyclin D were significantly reduced.The results showed that NaF in excess of 12 mg/kg blocked the cell cycle progression(this result was consistent with study in vitro),implying that splenic cell proliferation and its growth development were arrested.The decreased expression levels of cyclins were molecular basis of G0/G1 phase arrest.3.Effect of NaF on MAPK/ERK signaling pathwayFor Ras-Raf-MEK-ERK signaling pathway,when NaF exceeded 12 mg/kg,the m RNA and protein expression levels of Ras were significantly elevated.Also,the phosphorylated protein expression levels of Raf(B-Raf,C-Raf)were increased.Meanwhile,their m RNA expression levels were increased with a time and dose dependent manner at 21^st and 42^nd day of experiment.Additionally,the m RNA and protein levels of MEK1/2 were increased at the 21^stday of the experiment,while reduced at the 42^nd day.The ERK1/2 levels were significantly decreased at both 21^st and 42^nd day of the experiment.The above results indicated that NaF activated Ras to induce downstream Raf-MEK-ERK cascade reaction,but failed to activate ERK eventually,the proliferation signal from cell surface could not transmit to nucleus,interfering the regulation of cell proliferation,differentiation and meiosis.The G1 phase cell cycle was arrested,which ultimately suppressed splenic cell proliferation.EXP.3 Effects of NaF on splenic cell apoptosis in mice in vivoGroups and treatment of the experiment animals were consistent with Exp.2 in 1.The transmission electron microscopy(TEM)showed that splenic apoptotic lymphocytes were increased with a dose dependent manner,the mitochondria swelled,cristae fractured or even disappeared in NaF-treated groups.The FACs results showed that splenic apoptosis rate was increased with a time and dose dependent manner in NaF exceeded 12 mg/kg at21^st and 42^nd day of the experiment.Also,NaF in excess of 12 mg/kg caused a significant increase in ROS,and a decrease in MMP.At the 21^st and 42^nd day of the experiment,NaF-treatment induced ER stress,which was characterized by increasing m RNA and protein expression levels of endoplasmic reticulum stress markers GRP78 and GRP94.The UPR pathway responses detected that all the three UPR pathways were activated by NaF.The phosphorylated protein and m RNA expression levels of PERK?e IF2a?ATF4?IRE1?ATF6 and XBP1 were dramatic increased.At the 21^st and 42^nd day of the experiment,NaF reduced the m RNA and protein expression levels of anti-apoptotic Bcl-2 and Bcl-x L,increased the m RNA and protein expression levels of pro-apoptotic Bax and Bak.Also,the protein and m RNA expression levels of CHOP,caspase 12,caspase 3 and caspase 9 were increased significantly,which indicated that NaF activated CHOP and caspase 12 pathways via ER stress,ultimately induced apotosis.Above mentioned results demonstrated that NaF activated caspase 12 and CHOP via UPR of ER stress and induced splenic cell apoptosis.EXP.4 Effects of NaF on splenic cell autophagy in mice in vivoGroups and treatment of the experiment animals were consistent with Exp.2 in 1.The results of TEM and immunohistochemistry(IHC)showed that NaF increased autophagosomes or autolysosomes in the spleen.Simultaneously,the autophagy marker LC3 brown punctate staining was increased with a dose-and time-dependent manner.At the 21^st and 42^nd day of the experiment,the results of q RT-PCR and western blot showed that NaF inhibited m TOR activity,which was characterized by down-regulating m RNA and protein expression levels of PI3K,Akt and m TOR.Due to the suppression of m TOR activity,led to a significant increase of ULK1 and Atg13.Concurrently,the m RNA and protein expression levels of autophagy markers:LC3,Beclin1,Atg16L1,Atg12,Atg5were significant increased,p62 was decreased.The above-mentioned findings verified that NaF in excess of 12 mg/kg induced autophagy by inhibiting PI3K-Akt-m TOR signaling pathway.EXP.5 Effects of NaF on splenic immune function in mice in vivoGroups and treatment of the experiment animals were consistent with Exp.2 in 1.1.Effect of NaF on splenic adaptive immune functionAt the 21^st and 42^nd day of the experiment,the subpopulation results of T,B lymphocytes showed that NaF treatment reduced the percentages of CD3⁺,CD3⁺CD4⁺,CD3⁺CD8⁺and CD19⁺.NaF in excess of 12 mg/kg declined the m RNA and protein levels of IL-2?IL-6?IL-8?IL-1b?TGF-??TNF-??IFN-?as well as immunoglobulins(Ig A,Ig G and Ig M)contents,increased IL-4 and IL-10 m RNA and protein expression levels.Those results indicated that NaF in excess of 12 mg/kg reduced the splenic cellular and humoral immunity.2.Effect of NaF on splenic innate immune functionAt the 21^st and 42^nd day of the experiment,the results showed that NaF inactivated TLR2/My D88 signal,which were identified by prominently down-regulating m RNA and protein expression levels of TLR2,My D88,IRAK4,IRAK1,TRAF6,TAK1,MKK4/MKK7 and c-Jun,and up-regulating protein expression levels of p-JNK,p-MKK4,p-MKK7.These results implied that the TLR2-My D88 signaling pathway was inhibited.In summary,NaF in excess of 500?mol/L inhibited splenic lymphocytes proliferation in vitro.Therefore,a fluorosis model was established to investigate the effect and mechanism of NaF on splenic development,apoptosis,autophagy and immunity in mice in vivo.Results showed that NaF in excess of 12 mg/kg caused splenic histopathological lesions,G1 phase cell cycle arrest,ER stress,apoptosis,autophagy and immune dysfunction in spleen.Those above results were the molecular mechanism of NaF-induced splenic function damage.This research systematically investigated the effect and molecular mechanism of NaF on splenocytes and immune function by observing histological structure,cell proliferation,apoptosis,autophagy and immune response in mice.