Xiong Luo-Jie, Tian Yue-Feng, Xu Xiao-Shan, Zhai Chun-Tao, Li Wei
College of Acupuncture-Moxibustion, Tuina and Rehabilitation, Hunan University of CM, Changsha 410208, China.
Second Clinical College, Shanxi University of CM, Jinzhong 030619.
Zhongguo Zhen Jiu. 2023 May 12;43(5):529-36.
To observe the effects of herbal cake separated moxibustion on macrophage effector molecule T-cell immunoglobulin and mucin-domain containing-4 (Tim-4) and ubiquitination of programmed cell death protein 1 (PD-1) in rabbits with immunosuppression, and to explore the possible mechanism on herbal cake separated moxibustion in improving immunosuppression.
Thirty-two big-ear white rabbits were randomly divided into a normal group, a model group, a moxa stick moxibustion group and a herbal cake separated moxibustion group, 8 rabbits in each group. Except the normal group, the immunosuppression model was established by intraperitoneal injection of cyclophosphamide of60 mg/kg in the other 3 groups. "Shenque" (CV 8), "Shenshu" (BL 23), "Zusanli" (ST 36), etc. were selected in both the moxa stick moxibustion group and the herbal cake separated moxibustion group. Moxa stick moxibustion was applied in the moxa stick moxibustion group, one cone at each acupoint; herbal cake separated moxibustion was applied in the herbal cake separated moxibustion group, 5 cones at each acupoint. The intervention was given once every other day for 10 times in both groups. Leukocyte content in peripheral blood was detected by blood cell analyzer; the positive expression of PD-1 in CD T lymphocytes, CDT lymphocytes and CD macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD, CD and Tim-4 were detected by ELISA, and the expression of Tim-4 and F-box only protein 38 (FBXO38) in the liver and spleen tissues was measured by immunohistochemistry.
Compared with the normal group, in the model group, white blood cell count (WBC) and percentage of neutrophils (NEU%) were decreased while percentage of lymphocyte (LYM%) was increased (<0.01) in peripheral blood; the positive expression rates of PD-1 in CD T lymphocytes, CDT lymphocytes and CD macrophages in peripheral blood were increased (<0.01); the serum levels of IL-2, CD and Tim-4 were increased (<0.01), the serum level of CD was decreased (<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (<0.01). Compared with the model group, in the moxa stick moxibustion group and the herbal cake separated moxibustion group, WBC and NEU% were increased (<0.01); the positive expression rates of PD-1 in CD T lymphocytes, CDT lymphocytes and CD macrophages in peripheral blood were decreased (<0.01); the serum levels of IL-2, CD and Tim-4 were decreased (<0.01), the serum levels of CD were increased (<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (<0.01, <0.05). Compared with the moxa stick moxibustion group, in the herbal cake separated moxibustion group, the positive expression rate of PD-1 in CD macrophages in peripheral blood was increased (<0.05); serum level of Tim-4 was increased (<0.01); AOD of Tim-4 in the liver tissue was decreased (<0.05).
Herbal cake separated moxibustion can improve immunosuppression by regulating the expression of macrophage effector molecule Tim-4 and the FBXO38 mediated ubiquitination of PD-1, Tim-4 may be one of the specific indexes of immunomodulation involving with herbal cake separated moxibustion.
观察隔药饼灸对免疫抑制家兔巨噬细胞效应分子T细胞免疫球蛋白黏蛋白结构域分子4（Tim-4）及程序性细胞死亡蛋白1（PD-1）泛素化的影响，探讨隔药饼灸改善免疫抑制的可能机制。
将32只大耳白兔随机分为正常组、模型组、艾条灸组和隔药饼灸组，每组8只。除正常组外，其余3组腹腔注射60 mg/kg环磷酰胺建立免疫抑制模型。艾条灸组和隔药饼灸组均选取“神阙”（CV 8）、“肾俞”（BL 23）、“足三里”（ST 36）等穴位。艾条灸组采用艾条灸，每穴1壮；隔药饼灸组采用隔药饼灸，每穴5壮。两组均隔日干预1次，共干预10次。采用血细胞分析仪检测外周血白细胞含量；采用流式细胞术检测外周血CD⁺T淋巴细胞、CD⁺T淋巴细胞及CD⁺巨噬细胞中PD-1的阳性表达；采用酶联免疫吸附测定法（ELISA）检测血清白细胞介素2（IL-2）、CD、CD及Tim-4水平；采用免疫组织化学法检测肝和脾组织中Tim-4及F-box蛋白38（FBXO38）的表达。
与正常组比较，模型组外周血白细胞计数（WBC）、中性粒细胞百分比（NEU%）降低，淋巴细胞百分比（LYM%）升高（P<0.01）；外周血CD⁺T淋巴细胞、CD⁺T淋巴细胞及CD⁺巨噬细胞中PD-1阳性表达率升高（P<0.01）；血清IL-2、CD及Tim-4水平升高（P<0.01），血清CD水平降低（P<0.01）；肝组织中Tim-4及肝和脾组织中FBXO38的平均光密度（AOD）升高（P<0.01）。与模型组比较，艾条灸组和隔药饼灸组WBC及NEU%升高（P<0.01）；外周血CD⁺T淋巴细胞、CD⁺T淋巴细胞及CD⁺巨噬细胞中PD-1阳性表达率降低（P<0.01）；血清IL-2、CD及Tim-4水平降低（P<0.01），血清CD水平升高（P<0.01）；肝组织中Tim-4及脾组织中FBXO38的AOD降低（P<0.01，P<0.05）。与艾条灸组比较，隔药饼灸组外周血CD⁺巨噬细胞中PD-1阳性表达率升高（P<0.05）；血清Tim-4水平升高（P<0.01）；肝组织中Tim-4的AOD降低（P<0.05）。
隔药饼灸可能通过调节巨噬细胞效应分子Tim-4的表达及FBXO38介导的PD-1泛素化来改善免疫抑制，Tim-4可能是隔药饼灸免疫调节作用的特异性指标之一。
