Molecular mechanism of filiform needling and fire needling therapies in treatment of rat models of myelosuppression

doi:10.3760/cma.j.issn.1007-1245.2024.01.027

Abstract

Abstract:

Objective　To explore the molecular mechanism of filiform needling and fire needling in the treatment of myelosuppressive rat models. Methods　The study was from March to September 2022. Ninety-six SD rats who were 4-6 weeks old and whose body weight was 250-300 g were bought from Charles River. The rats were divided into a control group, a model group, a fire needle group, and a filiform needle group, with 24 in each group. Except the control group, the rats in the other groups were induced by cyclophosphamide (CTX) to establish the rat bone marrow suppression models. After the models were established successfully, the rats in the control group and the model group were grasped and fixed every day, but took no treatment. The rats in the fire needle group were fixed on the mouse boards in prone position according to the numbers, and took vertical acupuncture on their Zusanli points at 3 mm depth on both sides on the first, third, fifth, and seventh days after the modeling with the fire needles once every other day, for 7 consecutive days, and the needles were not left in the points. The rats in the filiform needle group were fixed on the mouse boards in prone position according to the numbers; the needles and acupoints were the same as those in the fire needle group; the needles were vertically punctured 3 mm into the points, and were left there for 6 min; the treatment started on the first day after modeling, once a day for 7 consecutive days. Ten μl blood was collected from the tail vein of the rats on the first to seventh days of intervention to measure the numbers of white and red blood cells and platelets, hemoglobin, and other blood routine indicators. The bone marrow tissue sections were made for for Wright's Giemsa staining. The flow cytometry was used to detect the bone marrow mononuclear fibroblast cycles. The expressions of cycle-related proteins in the bone marrow mononuclear fibroblasts were detected by Western blotting. LSD-t test, one-way analysis of variance, and Kruskal-Wallis test were applied. Results　The numbers of white blood cells and platelets in the model group were lower than those in the control group (all P<0.05), while the numbers of red blood cells and the contents of hemoglobin in the model group were higher than those in the control group (all P<0.05). One, five, and seven days after the treatment, the numbers of white blood cells [(3.53±0.19)×10⁹/L and (3.42±0.22)×10⁹/L vs. (3.12±0.10)×10⁹/L; (3.78±0.23)×10⁹/L and (3.65±0.29)×10⁹/L vs. (2.88±0.24)×10⁹/L; (4.08±0.43)×10⁹/L and (3.70±0.17)×10⁹/L vs. (2.27±0.12)×10⁹/L] and platelets [(79.72±3.89)×10⁹/L and (79.15±5.22)×10⁹/L vs. (70.17±2.20)×10⁹/L; (85.18±3.40)×10⁹/L and (81.88±4.97)×10⁹/L vs. (62.73±3.80)×10⁹/L; (92.18±7.64)×10⁹/L and (83.45±4.29)×10⁹/L vs. (51.92±1.64)×10⁹/L] in the fire needle group and the filiform needle group were higher than those in the model group (all P<0.05); the numbers of red blood cells [(1.36±0.03)×10¹²/L and (1.37±0.03)×10¹²/L vs. (1.52±0.03)×10¹²/L; (1.32±0.03)×10¹²/L and (1.34±0.02)×10¹²/L vs. (1.59±0.02)×10¹²/L; (1.24±0.07)×10¹²/L and (1.32±0.05)×10¹²/L vs. (1.68±0.03)×10¹²/L] and the contents of hemoglobin [(84.00±1.67) g/L and (84.33±1.86) g/L vs. (94.00±1.26)g/L; (82.00±2.68) g/L and (83.50±0.84) g/L vs. (97.83±1.17)g/L; (76.67±5.32) g/L and (81.83±3.43) g/L vs. (104.17±2.04) g/L] in the fire needle group and the filiform needle group were lower than those in the model group (all P<0.05). The numbers of bone marrow blast cells in the filiform needle group and the fire needle group were higher than that in the model group, and the rod-shaped nuclei cells were found. The percentages of bone marrow mononuclear fibroblasts in the G0/G1 phase were lower than those of the model group (all P<0.05); the change in the fire needle group was more significant than that in the filiform needle group, and the percentages of bone marrow mononuclear fibroblasts in the S phase and G2/M phase were always higher than those in the model group (all P<0.05). The relative expressions of CD4, CD6, Cylcin D1, and E2F1 proteins in the rat bone marrow mononuclear fibroblasts were significantly up-regulated in the filiform needle group and fire needle group 3 and 7 d after the treatment (all P<0.05). The expressions of cell cycle-related proteins, CD4, CD6, Cylcin D1, and E2F1, were more significantly up-regulated in the fire needle group. Conclusions　Filiform needling and fire needling both can improve cyclophosphamide-induced myelosuppression in rats. The molecular mechanism may be related to up-regulating the expressions of cell cycle-related proteins, CD4, CD6, Cylcin D1, and E2F1, in rat bone marrow mononuclear fibroblasts, thereby restoring the metabolic balance of proliferation and dormancy in bone marrow stromal cells.

Key words:

Myelosuppression, Filiform needle, Fire needle, Cell cycle, Cyclophosphamide

摘要：

目的　探讨毫针和火针治疗大鼠骨髓抑制模型的分子作用机制。方法　研究时间2022年3月至9月。采用随机数字表法将购于北京维通利华实验动物技术有限公司的96只4～6周龄、体质量为250～300 g的SD大鼠分为对照组、模型组、火针组和毫针组，各24只。除对照组外，其余各组大鼠用环磷酰胺诱导建立大鼠骨髓抑制模型，造模成功后，对照组和模型组大鼠每天陪同抓取、固定，但是不进行治疗；火针组大鼠根据编号依次将大鼠俯卧固定于鼠板上，选取双侧足三里穴于造模后第1天、第3天、第5天、第7天，采用火针点刺法治疗，火针垂直刺入3 mm，不留针，隔日1次，连续7 d；毫针组大鼠根据编号依次将大鼠俯卧固定于鼠板上，针具、选穴同火针组，垂直刺入3 mm，留针6 min，于造模后第1天开始，每天1次，连续7 d。分别于干预1～7 d取大鼠尾静脉血10 μl测定白细胞数量、红细胞数量、血红蛋白含量和血小板数量等血常规指标；制作骨髓组织切片，进行瑞氏吉姆沙染色；流式细胞术检测骨髓单核成纤维细胞周期；免疫印迹法检测骨髓单核成纤维细胞周期相关蛋白的表达。采用LSD-t检验、单因素方差分析和Kruskal-Wallis检验。结果　模型组大鼠白细胞数量和血小板数量均低于对照组（均P<0.05），红细胞数量、血红蛋白含量高于对照组（均P<0.05）。治疗后3、5、7 d，火针组和毫针组白细胞数量［（3.53±0.19）×10⁹/L、（3.42±0.22）×10⁹/L比（3.12±0.10）×10⁹/L；（3.78±0.23）×10⁹/L、（3.65±0.29）×10⁹/L比（2.88±0.24）×10⁹/L；（4.08±0.43）×10⁹/L、（3.70±0.17）×10⁹/L比（2.27±0.12）×10⁹/L］和血小板数量［（79.72±3.89）×10⁹/L、（79.15±5.22）×10⁹/L比（70.17±2.20）×10⁹/L；（85.18±3.40）×10⁹/L、（81.88±4.97）×10⁹/L比（62.73±3.80）×10⁹/L；（92.18±7.64）×10⁹/L、（83.45±4.29）×10⁹/L比（51.92±1.64）×10⁹/L］高于模型组（均P<0.05），红细胞数量［（1.36±0.03）×10¹²/L、（1.37±0.03）×10¹²/L比（1.52±0.03）×10¹²/L；（1.32±0.03）×10¹²/L、（1.34±0.02）×10¹²/L比（1.59±0.02）×10¹²/L；（1.24±0.07）×10¹²/L、（1.32±0.05）×10¹²/L比（1.68±0.03）×10¹²/L］和血红蛋白含量［（84.00±1.67）g/L、（84.33±1.86）g/L比（94.00±1.26）g/L；（82.00±2.68）g/L、（83.50±0.84）g/L比（97.83±1.17）g/L；（76.67±5.32）g/L、（81.83±3.43）g/L比（104.17±2.04）g/L］低于模型组（均P<0.05）；毫针组和火针组中骨髓原始细胞数量高于模型组，且可见杆状核细胞的出现；火针组和毫针组G0/G1期骨髓单核成纤维细胞百分率始终低于模型组（均P<0.05），其中火针组变化较毫针组更显著，并且S期和G2/M期骨髓单核成纤维细胞百分率始终高于模型组（均P<0.05）。毫针组和火针组治疗后3、7 d大鼠骨髓单核成纤维细胞中CD4、CD6、Cylcin D1和E2F1蛋白相对表达量明显上调（均P<0.05）。火针组细胞周期相关蛋白CD4、CD6、Cylcin D1和E2F1的表达上调更显著。结论　毫针和火针治疗均可以改善环磷酰胺诱导的大鼠骨髓抑制，其分子机制可能与上调大鼠骨髓单核成纤维细胞中细胞周期相关蛋白CD4、CD6、Cylcin D1和E2F1表达有关，从而恢复骨髓基质细胞增殖和休眠的代谢平衡。

关键词:

骨髓抑制, 毫针, 火针, 细胞周期, 环磷酰胺

Yang Xiaolin, Li Yan.

Molecular mechanism of filiform needling and fire needling therapies in treatment of rat models of myelosuppression [J]. International Medicine and Health Guidance News, 2024, 30(1): 130-136.

杨晓琳李岩.

毫针和火针疗法治疗骨髓抑制大鼠模型的分子作用机制 [J]. 国际医药卫生导报, 2024, 30(1): 130-136.

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