The Protective Effect And Mechanism Of Ellagic Acid On Immune Damage Induced By Beryllium Sulfate In Rats

Objective:To investigate the protective effect and mechanism of ellagic acid（EA）on immune damage induced by beryllium sulfate（BeSO₄）in SD rats.Methods:（1）Forty SPF-grade male Sprague-Dawley rats were randomly divided into four equal groupscontaining ten rats as follows:control group was treated by intratracheal instillation with sterile distilled water（1m L/kg·BW）followed by gavage with 1%carboxymethyl cellulose（10m L/kg·BW per day）for 8 weeks;Group 2（BeSO₄ group）was treated by intratracheal instillation with BeSO₄（12 mg/kg·BW）followed by gavage with 1%carboxymethyl cellulose（10 m L/kg·BW per day）for 8 weeks;Group 3（BeSO₄+EA 100 mg/kg·BW group）and 4（BeSO₄+EA 300mg/kg·BW group）were treated by intratracheal instillation with BeSO₄（12 mg/kg·BW）followed by gavage with EA at dose of 100 and 300mg/kg·BW per day for 8 weeks.（2）Observation of body weight and general condition:Body weights,food and water intakes were measured every day.In addition,the changes of coat,behavior and mental status were recorded.（3）At the end of 6^th week and 8^th week,5 rats in each group were randomly selected,anesthetized with ether,and killed by bleeding through the abdominal aorta.Blood sample was collected for hematologic parameters measurement.The lymphocyte subsets（CD3⁺,CD4⁺,CD8⁺）in peripheral blood were detected by flow cytometry.The levels of immunoglobulin IgA and Ig G in serum were measured by ELISA.（4）The spleen coefficient was calculated as organ weight/body weight;For the histopathological observation,spleenswere fixed in 10%neutral formalin solution,and stained with hematoxylin and eosin（H&E）for microscopic examination.For the splenocyte proliferation analysis,splenocytes were isolated from spleen.Afterwards,splenocytes were suspended in RPMI-1640 complete medium,seeded into 96-well plates at5×10⁵ cells/well and incubated with Con A or LPS.The content of malondialdehyde（MDA）and the activity of superoxide dismutase（SOD）in spleen were determined by thiobarbituric acid method and nitro-blue tetrazolium method.The m RNA expressions and protein levels of bax,bcl-2,Caspase-3 and PARP in spleen were detected by q RT-PCR and western blotting.Results.（1）Compared to the control group,the rats in the BeSO₄-intoxicant group exhibited decreased activity,reduced food and water consumptions,and mental depression.Administration of EA recovered rats towards normal.（2）Compared to the control group,rats in the BeSO₄ group showed lower body weight and higher spleen organ index（P<0.01）;When compared to the BeSO₄ group,treatment of EA caused elevation in body weight,and alleviation in spleen organ index（P<0.05）.Our evaluations showed a thickening of the marginal area,localized central dilatation of the spleen nodules,red pulp with blood stasis,and nuclear fragmentation in spleen sections of the BeSO₄-intoxicant rats.Administration of EA significantly mitigated these histopathological symptoms.（3）Compared to the control group,rats in the BeSO₄ group showed significantly higher WBC,Neu,Lym,and PLT,respectively.Treatment with EA at dose of 100 and 300 mg/kg/day caused significant decreases in above-mentioned variables compare to those of the BeSO₄-intoxicant rats.（4）Compared to the control group,Con A and LPS signifificantly increased the relative cell viability of splenocyte in the BeSO4 group（P<0.01）,whereas treatment of EA significantly reduced the Con A-stimulated splenocyte proliferation and the LPS-stimulated splenocyte proliferation when compared to the BeSO4 group（P<0.01）.（5）Compared to the control group,rats in the BeSO₄ group showed increased number of CD4⁺cells and decreased number of CD8⁺cells,resulting in a significant elevation in CD4⁺/CD8⁺cell subset ratio（P<0.01）.Administration of EA caused significant decreases in the number of CD4⁺cells and CD4⁺/CD8⁺cell subset ratio,compared to those of the BeSO₄ group（P<0.01）.（6）The levels of IgA and Ig G showed significant decrease in the BeSO₄ group when compared to those of the control group（P<0.05）.Administration of EA significantly increased the levels of IgA and Ig G,compared to those of the BeSO₄ group（P>0.05）.（7）Compared with the control group,treatment of BeSO₄ reduced the SOD activity.Administration of EA restored the activity of SOD,The MDA content increased in the BeSO₄-intoxicant rats when compared to the control group,whereas administration of EA significantly alleviated MDA content in the spleen tissue of BeSO₄-intoxicant rats.（8）BeSO₄ has significantly increased apoptosis as evidenced by increased expression of apoptotic bax,caspase-3,PARP and decreased expression of bcl-2 markers respectively.Obviously,expression of pro-apoptotic bax,caspase-3 and PARP were decreased,while expression of anti-apoptotic bcl-2 was increased after EA administration.Conclusions:（1）BeSO₄ could cause structural damage and immune functional damage in the spleen of SD rats.（2）Ellagic acid against structural damage and immune functional damage in spleen induced by BeSO₄may through inhibition of oxidative stress and apoptosis.