麦粒灸对骨髓抑制小鼠骨髓细胞Wnt/β-catenin信号通路的影响

Effect of wheat-grain moxibustion on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition

目的:观察麦粒灸“大椎”“足三里”“三阴交”对骨髓抑制小鼠骨髓细胞Wnt/β-连环蛋白(β-catenin)信号通路相关蛋白表达的影响,探讨麦粒灸治疗骨髓抑制的可能机制。方法:将45只SPF级雄性CD1(ICR)小鼠随机分为空白组、模型组和麦粒灸组,每组15只。采用腹腔注射环磷酰胺(CTX,80 mg/kg)制备骨髓抑制模型。麦粒灸组于“大椎”“足三里”“三阴交”行麦粒灸治疗,每穴3壮,每壮约30 s,每日1次,连续7 d。检测小鼠造模前、干预前及干预3、5、7 d后白细胞计数(WBC)。干预后,观察小鼠一般情况;检测小鼠骨髓有核细胞计数;采用ELISA法检测小鼠血清白细胞介素-3(IL-3)、白细胞介素-6(IL-6)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)的含量;采用Western blot法和实时荧光定量PCR法检测小鼠骨髓细胞β-catenin、细胞周期蛋白D1(Cyclin D1)、增殖调控蛋白(C-Myc)蛋白及mRNA表达。结果:与空白组比较,模型组小鼠反应迟钝、步态不稳、体重减轻,WBC、骨髓有核细胞计数及血清IL-3、IL-6、GM-CSF含量减少(P<0.01),骨髓细胞β-catenin、CyclinD1、C-Myc蛋白及mRNA表达减少(P<0.01);与模型组比较,麦粒灸组小鼠一般情况好转,WBC、骨髓有核细胞计数及血清IL-3、IL-6、GM-CSF含量增多(P<0.01,P<0.05),骨髓细胞β-catenin、Cyclin D1、C-Myc蛋白及mRNA表达增加(P<0.05)。结论:麦粒灸对骨髓抑制具有治疗作用,其作用机制可能是通过激活骨髓细胞Wnt/β-catenin信号通路,改善骨髓造血微环境,促进骨髓细胞增殖。

Objective To observe the effect of wheat-grain moxibustion at "Dazhui"(GV 14), "Zusanli"(ST 36) and "Sanyinjiao"(SP 6) on Wnt/β-catenin signaling pathway in bone marrow cell in mice with bone marrow inhibition, and to explore the possible mechanism of wheat-grain moxibustion in treating bone marrow inhibition. Methods Forty-five SPF male CD1(ICR) mice were randomly divided into a blank group, a model group and a wheat-grain moxibustion group, 15 mice in each group. The bone marrow inhibition model was established by intraperitoneal injection of 80 mg/kg of cyclophosphamide(CTX). The mice in the wheat-grain moxibustion group were treated with wheat-grain moxibustion at "Dazhui"(GV 14), "Zusanli"(ST 36) and "Sanyinjiao"(SP 6), 3 moxa cones per acupoint, 30 s per moxa cone, once a day, for 7 consecutive days. The white blood cell count(WBC) was measured before modeling, before intervention and 3, 5 d and 7 d into intervention. After intervention, the general situation of mice was observed;the number of nucleated cells in bone marrow was detected;the serum levels of interleukin-3(IL-3), interleukin-6(IL-6) and granulocyte macrophage colony stimulating factor(GM-CSF) were measured by ELISA;the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc in bone marrow cells was measured by Western blot and real-time PCR method. Results Compared with the blank group,the mice in the model group showed sluggish reaction, unstable gait, decreased body weight, and the WBC, number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were decreased(P<0.01), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was decreased(P<0.01). Compared with the model group, the mice in the wheat-grain moxibustion group showed better general condition, and WBC, the number of nucleated cells in bone marrow as well as serum levels of IL-3, IL-6, GM-CSF were increased(P<0.01, P<0.05), and the protein and mRNA expression of β-catenin, cyclinD1 and C-Myc was increased(P<0.05). Conclusion Wheat-grain moxibustion shows therapeutic effect on bone marrow inhibition, and its mechanism may be related to activating Wnt/β-catenin signaling pathway in bone marrow cells, improving bone medullary hematopoiesis microenvironment and promoting bone marrow cell proliferation.