| Objective:(1) To discuss the Double-reinforcing and One-unblocking acupuncture method whether could delay the immunosenescence course in aging model rats induced by D-galactose;(2) To discuss the improvement of the Double-reinforcing and One-unblocking acupuncture method on the vaccine response in aging model rats induced by different concentrations of DTaP vaccine;(3) To test the involved indexes of aging model rats induced by D-galactose, and to identify the models are performed;(4) To use the Double-reinforcing and One-unblocking acupuncture method on both aging model rats and normal rats, to discuss acupuncture whether could improve the immune effect on rats and could be used as adjuvant, moreover, to discuss acupuncture whether could delay the immunosenescence course in aging model rats.Methods:(1)40SD rats, male only,30rats were selected randomly to make the ageing model rats by injected subcutaneously with D-galactose for42d, and were divided into3groups:double reinforcing-one unblocking acupuncture group (GuanyuanCV4, HousanliST36and BaihuiGV20points, used electro-acupuncture therapy on GuanyuanCV4and HousanliST36only); acupuncture control group (WeizhongBL40, ShuifenCV9and YintangGV29, used electro acupuncture therapy on WeizhongBL40and ShuifenCV9only);ageing model group, then, the last10rats were in normal control group. With3-week treatment, spleen index and thymus index were observed; and the percentage of CD8+T/T cell and the percentage of CD8+CD28-T/CD8+T cell were test by flow cytometer.(2)Wistar female rats48was randomly divided into6groups, namely:1/10vaccine immune acupuncture group;1/10vaccine immune control group;1/20vaccine immune acupuncture group;1/20vaccine immune control group;1/40vaccine immune acupuncture group;1/40vaccine immune control group. Every group were injected with DTaP vaccine, acupuncture groups were treated with electro-acupuncture therapy and moxibustion on BaiHuiGV20HousanliST36and GuanyuanCV4for3-week. At5th week, all rats were collected whole blood, serum separation, diphtheria endotoxins antibody titer were detected by Vero cell separation. The spleen lymphocyte specific proliferation index was tested by MTS colorimetric method, CD4+T/CD8+T lymphocyte ratio was tested by flow cytometer and the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method(compared with GAPDH)(3)Wistar rats20, female only,10rats were randomly selected to make aging model by injected subcutaneously with D-galactose for42d, the rest is in normal control group. After the Morris water maze, the serum SOD activity, the serum lipid, TG and the GHOL level were tested, the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method.(4)Wistar rats48, female only, all rats were randomly divided into4groups, namely:model immune acupuncture and moxibustion group; immune model control group; normal immune acupuncture and moxibustion group and normal immune control group. Normal saline was injected subcutaneously to rats in the normal immune group, and D-galactose was injected subcutaneously to rats in the immune model group for6weeks. At7th week, all rats were injected with DTaP vaccine, and treated with electro-acupuncture stimulation and moxibustion on GuanyuanCV4, HousanliST36and BaihuiGV20points for3-week expect control groups. After feed for2weeks, all rats were collected whole blood, serum separation, diphtheria endotoxins antibody titer were detected by Vero cell separation; The blood lipid, protein level and the activity of serum SOD were tested; CD4+/CD8+T cell ratio was tested by flow cytometer and the relative expression of splenocyte HSP70mRNA was detected by RT-PCR method.Results:(1)Compare with normal control group, in ageing model group, spleen index and thymus index were lower (P<0.05); the percentage of CD8+CD28T/CD8+T cell were higher remarkably (P<0.01); the percentage of CD8+T/T cell were higher remarkably (P<0.01). Compare with ageing model group, in double reinforcing-one unblocking acupuncture group, spleen index and thymus index increased (P<0.05); the percentage of CD8+CD28-T/CD8+T cell decreased remarkably (P<0.01). Compare with ageing model group, in acupuncture control group, the percentage of CD8+CD28-T/CD8+T cell decreased remarkably too(P<0.01). However, the percentage of CD8+CD28-/CD8+T cell in double reinforcing-one unblocking acupuncture group decreased much more significantly.(2) Compared with1/20vaccine immune control group, the antibody titer in1/20vaccine immune acupuncture group is significantly higher (P<0.01); the antibody titer in1/10vaccine immune acupuncture group and1/10vaccine immune controls both are higher, and there was no significant difference between each other; the antibody titer of1/40vaccine immune acupuncture group and1/40vaccine immune control group have no significant difference. The splenocyte proliferation experiment found vaccine is toxic for cells and is not suitable for the specific proliferation experiment. Compared with control group, the percentage of CD4+T/CD8+T cell in1/20vaccine immune acupuncture group is increased slightly(P>0.05); the relative expression of spenocyte HSP70mRNA in1/20vaccine immune acupuncture group detected by RT-PCR increased slightly (P>0.05)(3)Compare with normal control group, the explore space and search latency of aging model rats in water maze test shortened remarkably (P<0.01); serum SOD activity decreased significantly (P <0.01); the TG and the GHOL level increased (P<0.05); the relative expression of splenocyte HSP70mRNA decreased (P<0.05).(4)Compare with normal immune control group, in model immune control group, SOD activity decreased remarkably (P<0.01), TG and CHOL level increased (P<0.05); TP and GLB decreased (P<0.05); antibody titer decreased significantly (P<0.01); CD4+T/CD8+T cell decreased remarkably (P<0.01); the relative expression of spenocyte HSP70mRNA decreased remarkably (P<0.01). Compare with model immune control group, in immune model acupuncture and moxibustion group, TG and CHOL decreased (P<0.05); TP and GLB increased (P<0.05); SOD activity increased (P<0.05); antibody titer increased significantly (P<0.01); CD4+T/CD8+T increased (P <0.01); the relative expression of spenocyte HSP70mRNA expression increased (P<0.01). Compare with normal immune control group, in normal immune acupuncture and moxibustion group, TG and CHOL decreased (P<0.05); TP and GLB increased (P<0.05); antibody titer increased (P<0.05).Conclusion:(1)Electro-acupuncture treatment can delay immunosenescence course in aging model rats induced by D-galactose, by improving of spleen index and thymus index, and regulating the proportion of T cell subgroup.(2) Acupuncture and moxibustion treatment could be a new adjuvant, by enhancing the vaccine response in rats, and the optimum concentration is1/20.(3) Subcutaneous injection of D-galactose can impair the learning and memory in rats, increase the blood lipid, reduce HSP70mRNA expression, thus it is a better method on the construction of aging model rats.(4) Probably based on increase of HSP70mRNA level, acupuncture and moxibustion treatment could improve the involved indexes, and enhance the immune effect in humoral immunity and cellular immunity on aging rats.To sum up, according to induce the HSP70expression, acupuncture and moxibustion treatment can delay immunosenescence, improve the involved indexes, and enhance the immunity effect in humoral immunity and cellular immunity on aging rats and normal rats, and as vaccine adjuvant in the process. |
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