| Objective:Observe the effects of nourishing kidney Yin method of aging rats in immune function and caspase related protease by modern experimental way and analyze the effect of nouring Yin method in anti-aging primarily,to explore its mechanism from possible angles.Methods:1.Groups:40 healthy Wistar rats were randomly divided into four groups,Normal group(control group /N group),model group(control group/M group),positive control group(vitamin E group/P group),Nourishing kidney Yin method group(Liuweidihuang Pill group/NK group)with 10 rats in each group.2.Modeling and administration:Group M,group P and group NK was given D-gal 100mg/kg?d induced senescence by intraperitoneal injection at the same time,group P was given vitamin E 0.027g/kg?d orally,group NK was given Liuweidihuang pill solution 3.24g/kg?d orally,group N and group M were given the same volume of Normal saline.3.Outcome measures and detection methods:Obserbed the general state and weight of the rats weekly,adjusting the dosage according to body weight.After 8 weeks,two hours after the last dose,Exsanguinate the orbital blood 1.5ml of rats,0.5ml were used for rat urea nitrogen(BUN),serum creatinine(Scr)by automatic biochemical analyzer.1ml blood application for NO and NOS values with enzyme nitrate reduction method(Griess).After anesthetized and executed,the thymus and spleen of rats were peeling out,weighed which percentage body weight were immune organ index.Take a side of the rats' kidney cortex for immunohistochemistry: First,paraffin-embeddedtissue,sliced,dewaxing,EDTA antigen retri-eval solution cartridge BOC antigen retrieval,then sliced into 3% hydrogen peroxide solution to block endogenous peroxidase,3% BSA closed after joining Configuring a good anti-Bcl-2?Bax and Caspase-3,and then the DAB color-stained nucleus,and finally dehydrated,mounted with neutral gum,parallel microscope microscopic examination,image acquisition.apoptotic protein imaging and intensity in rats kidny tissue in every group were evaluated differentiated by immunohistochemical score evaluation.Normality and homogeneity of variance between each groups were used in statistical analysis,then using analysis of variance,pairwise comparisons using LSD-t test.Results:1.Generally,the rats in group N had good appetie,bright and thick fur,lively and difficult to capture.Contrary,the rats in group M Model rats gradually appeared chlorotic fur,loose stoole,Diet significantly reduced,running and crawling slowly,slow response and so on what symptons just like the natural aging rats.The performance in intervention group between the groups above.The body weight between groups were statistically differences in 3rd week(P<0.05),then there are significant differences between the groups in 6th weeks(P<0.01),and group P(328.30±8.23)compared with group NK(350.50±16.75)had difference(P<0.05);To the end of the 8th week,a significant difference between the groups was observed(P<0.01),group P(361.80 ± 13.44)and group N(375.80 ±5.87),group NK(371.45 ± 8.48)had difference(P<0.05),group M(345.30±12.42)and group N,group P and group NK had significant differences(P<0.01),what prompted rats in experiment fed properly,D-gal induced aging rat models were successfully manufactured,vitamin E and Liuweidihuang pills had a certain anti-aging effects,and the effects of Liuweidihuang pill is superior than vitamin E.2.The comparision of rats renal function after medication in every groups:There were significant differences in each group on renal blood urea nitrogen(BUN)and serum creatinine(Scr)(P<0.01).According to pairwise comparison that group M(17.3 ± 4.12,101.12 ± 25.67)and group N(7.34 ± 0.55,33.75 ± 5.61),group M and group P(13.1 ± 2.54,87.58 ± 18.97),group M and group NK(12.5 ± 2.31,85.67 ± 17.65)were significantly different on BUN and Scr(P<0.01).While group N and group P,group NK had differences in those areas too(P<0.05),group P and group NK had no significant differences.3.Immunologic function: 3.1 At aspect of immune organ index,the thymus indexes in every group were different after 8 weeks(P<0.05),and the pairwise comparison found that group N(1.34 ± 0.19)and group M(0.99 ± 0.27)were significantly different(P<0.05).Spleen indexes were significant different(P<0.05),while group M(1.67 ± 0.44)and group N(2.19 ± 0.68)had significant difference(P<0.01),group M and group NK(2.06 ± 0.52)had difference(P<0.05).3.2 The comparision of rats serum NO,NOS in every group:NO were significant difference in each group(P<0.01).Among them,group M(253.5 ± 31.8)significantly increased compared with group N(154.3 ± 32.8),group P(192.5 ± 35.6)(P<0.01)and increased compared with group NK(216.5 ± 32.2)(P<0.05).Compared with the group N,group P and group NK also increased statistically(P<0.05),whereas no significant difference between group P and group NK(P> 0.05).NOS in each group had statistically difference(P<0.05).Among them,group M(44.4±6.7)significantly increased compared with group N(29.8 ± 5.6),group P(32.6 ± 4.8)(P<0.01)and increased compared with group NK(33.2 ± 5.1)(P<0.05).Compared with group N,group P and group NK also increased statistically(P<0.05),whereas there was no significant difference between group P and group NK(P>0.05).4.The comparision of rats Bcl-2?Bax and Caspase-3 in each group: Bcl-2 expressed more in group N which had more positive cells,compared with other groups,and the cells were stained dark,there was a statistically significant difference(P<0.01)in scores,while there was no significant difference between group N and group NK(P>0.05).Bax expressed more in group M which had more positive cells,compared with other groups,and the cells were stained dark,there was a statistically significant difference(P<0.01).However there was no statistically difference between group NK and group P(P>0.05).Caspase-3 expressed more in group M which had more positive cells,compared with other groups,and the cells were stained dark,there was a statistically significant difference(P<0.01).While there was statistically difference between group NK and group P(P<0.05).Conclusion:Compared with normal rats that aging rats reduced in body weight,renal function and immune organ index.In contrast,secretion of NO and NOS and apoptosis increased.But Liuweidi-huangwan Pill as the representative of nourishing kidney Yin method could delay the decrease in rats body weight,renal function and immune organ index,and inhibit the excessive secretion of NO and NOS and apoptosis.Thus Nourishing kidney Yin method may have some effect on the immune function of aging rats,through this process may delay the aging process. |
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