细辛脂素对心脏移植大鼠术后存活时间和大鼠脾脏和外周血免疫排斥作用研究

Effect of Asarinin on survival time after heart transplantation and anti-immune rejection of spleen and peripheral blood in rats

目的探究细辛脂素对同种异体异位心脏移植术后大鼠移植心脏存活时间的影响及其在脾脏及外周血的抗免疫排斥作用。方法以64只Wistar大鼠为供体,64只SD大鼠为受体,建立大鼠同种异体异位心脏移植模型。移植成功后用简单随机分组法将64只大鼠分为对照组、环孢素A组、细辛脂素组及半量环孢素A+半量细辛脂素组,每组16只。环孢素A组以环孢素A 5 mg/kg灌胃;细辛脂素组以细辛脂素25 mg/kg灌胃;半量组以环孢素A 2.5 mg/kg+细辛脂素12.5 mg/kg灌胃;对照组以同等容积生理盐水灌胃。给药1周后,其中一半大鼠用于观察存活时间,另一半大鼠水合氯醛充分麻醉后,取脾脏的一半用显微镜下观察切片形态学变化;另一半脾脏用RT-PCR检测IFN-γ和IL-4表达;流式细胞术检测外周血共刺激分子CD80、CD86、CD40的表达。结果对照组移植心脏存活时间为(8.4±0.9)d,环孢素A组为(30.5±8.3)d,细辛脂素组为(16.5±4.3)d,半量组为(26.1±5.2)d,与对照组比较各组移植心脏存活时间均延长,且差异均有统计学意义(P均<0.05)。脾脏组织HE染色提示与对照组比较,各组损伤均减轻;对照组脾脏IFN-γ相对表达量为1.055±0.083,环孢素A组为0.396±0.038,细辛脂素组为0.833±0.094,半量组为0.862±0.104,后三组与对照组比较均降低,且差异均有统计学意义(P均<0.05)。对照组IL-4相对表达量为1.429±0.234,环孢素A组为3.808±0.729,细辛脂素组为2.209±0.306,半量组为2.323±0.321,后三组与对照组比较均上升,且差异均有统计学意义(P均<0.05)。对照组外周血CD80、CD86、CD40表达量分别为98.21±0.54、85.78±0.89和96.36±0.66,环孢素A组为89.26±0.36、56.86±2.32和88.11±1.61,细辛脂素组为94.19±0.47、79.01±1.12和87.86±1.67,半量组为94.87±0.74、80.81±0.98和89.71±0.97,后三组与对照组比较均降低,且差异均有统计学意义(P均<0.05)。结论细辛脂素可以延长大鼠移植心脏的存活时间,抑制脾脏组织的免疫性损伤,降低脾脏中IFN-γ的表达,增加脾脏中IL-4的表达,抑制外周血共刺激分子CD80、CD86、CD40的表达。

Objective To investigate the effect of Asarinin on the survival time of transplanted heart after allogeneic heterotopic heart transplantation and to further verify the anti-immune rejection effect of Asarinin in spleen and peripheral blood.Methods Using 64 Wistar rats as donors,64 SD rats as recipients to establish the allogeneic heterotopic heart transplantation model in rats.After successful transplantation,64 rats were use simple randomization divided into control group,cyclosporine A(CsA)group,Asarinin group and half CsA+half Asarinin group with 16 rats in each group.CsA group was given 5 mg/kg by gavage;Asarinin group was given 25 mg/kg;half dose group was given CsA 2.5 mg/kg+Asarinin 12.5 mg/kg and the control group was given the same volume of normal saline by gavage.After administration for 1 week,half of them were used to observe the survival time.The other half of the rats were fully anesthetized with chloral hydrate,spleen and peripheral blood were taken.Half of the spleen was taken to observe the slices under the microscope.The other half of spleen was used RT-PCR to detect the relative expression of IFN-γand IL-4.The expression of co-stimulatory molecules CD80,CD86 and CD40 in peripheral blood were detected by flow cytometry.Results Survival time of transplanted heart was control group(8.4±0.9),CsA group(30.5±8.3),Asarinin group(16.5±4.3)and half-dose group(26.1±5.2)days.Compared with control group,survival time of heart transplantation became prolonged in all groups and the difference was statistically significant(P<0.05).HE staining of splenic tissue showed that,as compared with control group,the injury of each group was alleviated.The relative expression of IFN-γin spleen was control group(1.055±0.083),CsA group(0.396±0.038),Asarinin group(0.833±0.094)and half-dose group(0.862±0.104).The last three groups were lower than control group and the difference was statistically significant(P<0.05).The relative expression of IL-4 in spleen was control group(1.429±0.234),CsA group(3.808±0.729),Asarinin group(2.209±0.306)and half-dose group(2.323±0.321).The last three groups all spiked as compared with control group and the difference was statistically significant(P<0.05).The expressions of CD80,CD86 and CD40 in peripheral blood were control group(98.21±0.54),(85.78±0.89)and(96.36±0.66),CsA group(89.26±0.36),(56.86±2.32)and(88.11±1.61),Asarinin group(94.19±0.47),(79.01±1.12)and(87.86±1.67)and half-dose group(94.87±0.74),(80.81±0.98)and(89.71±0.97)respectively.The last three groups were lower than control group and the difference was statistically significant(P<0.05).Conclusions Asarinin can prolong the survival time of transplanted heart after allogeneic heterotopic heart transplantation in rats,inhibit the immune injury of spleen after allogeneic heterotopic heart transplantation in rats,decrease IFN-γin spleen,increase IL-4 in spleen and inhibit the expression of peripheral blood costimulatory molecules CD80,CD86 and CD40.