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Title: [Effects of herbal cake separated moxibustion on Tim-4 and ubiquitination of PD-1 in rabbits with immunosuppression].
Author: Xiong LJ, Tian YF, Xu XS, Zhai CT, Li W.
Journal: Zhongguo Zhen Jiu; 2023 May 12; 43(5):529-36. PubMed ID: 37161806.
Abstract:
OBJECTIVE: 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. METHODS: 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⁺₄ T lymphocytes, CD⁺₈T lymphocytes and CD⁺₆₈ macrophages in peripheral blood was measured by flow cytometry, the serum levels of interleukin 2 (IL-2), CD₈, CD₆₈ 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. RESULTS: 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 (P<0.01) in peripheral blood; the positive expression rates of PD-1 in CD⁺₄ T lymphocytes, CD⁺₈T lymphocytes and CD⁺₆₈ macrophages in peripheral blood were increased (P<0.01); the serum levels of IL-2, CD₆₈ and Tim-4 were increased (P<0.01), the serum level of CD₈ was decreased (P<0.01); the average optical density (AOD) of Tim-4 in the liver tissue and FBXO38 in the liver and spleen tissues was increased (P<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 (P<0.01); the positive expression rates of PD-1 in CD⁺₄ T lymphocytes, CD⁺₈T lymphocytes and CD⁺₆₈ macrophages in peripheral blood were decreased (P<0.01); the serum levels of IL-2, CD₆₈ and Tim-4 were decreased (P<0.01), the serum levels of CD₈ were increased (P<0.01); the AOD of Tim-4 and FBXO38 in the liver tissue and FBXO38 in the spleen tissue was decreased (P<0.01, P<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⁺₆₈ macrophages in peripheral blood was increased (P<0.05); serum level of Tim-4 was increased (P<0.01); AOD of Tim-4 in the liver tissue was decreased (P<0.05). CONCLUSION: 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. 目的：观察隔药饼灸对免疫抑制兔巨噬细胞效应分子T细胞免疫球蛋白黏蛋白分子-4（Tim-4）及细胞程序性死亡受体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。方法：将32只大耳白兔随机分为空白组、模型组、艾条灸组及隔药饼灸组，每组8只。除空白组外，其余3组均以60 mg/kg剂量腹腔注射环磷酰胺制备免疫抑制模型。艾条灸组予艾条灸，每穴灸1壮；隔药饼灸组予隔药饼灸，每穴灸5壮。均选取“神阙”“肾俞”“足三里”等穴，隔日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；流式细胞术检测外周血PD-1在CD⁺₄、CD⁺₈T淋巴细胞及CD⁺₆₈巨噬细胞中的阳性表达；ELISA法检测血清白细胞介素2 （IL-2）、CD₈、CD₆₈及Tim-4含量；免疫组化法检测肝脏、脾脏Tim-4及F-box蛋白38（FBXO38）表达。结果：与空白组比较，模型组外周血白细胞计数（WBC）及中性粒细胞百分比（NEU%）降低、淋巴细胞百分比（LYM%）升高（P<0.01）；外周血PD-1在CD⁺₄、CD⁺₈T淋巴细胞及CD⁺₆₈巨噬细胞中的阳性表达率升高（P<0.01）；血清IL-2、CD₆₈及Tim-4含量升高（P<0.01），CD₈含量降低（P<0.01）；肝脏Tim-4及肝脏、脾脏FBXO38平均吸光度值增高（P<0.01）。与模型组比较，艾条灸组与隔药饼灸组WBC及NEU%升高（P<0.01）；外周血PD-1在CD⁺₄、CD⁺₈T淋巴细胞及CD⁺₆₈巨噬细胞中的阳性表达率降低（P<0.01）；血清IL-2、CD₆₈与Tim-4含量降低（P<0.01），CD₈含量升高（P<0.01）；肝脏Tim-4、FBXO38及脾脏FBXO38平均吸光度值降低（P<0.01，P<0.05）。与艾条灸组比较，隔药饼灸组外周血PD-1在CD⁺₆₈巨噬细胞中的阳性表达率升高（P<0.05）；血清Tim-4含量增高（P<0.01）；肝脏Tim-4平均吸光度值降低（P<0.05）。结论：隔药饼灸可通过调节巨噬细胞效应分子Tim-4表达及FBXO38介导的PD-1泛素化改善免疫抑制状态，Tim-4可能是其参与免疫调控的特异性指标之一。.

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