Objective: To observe the anti-tumor effect of moxibustion with seed-sized moxa cones on Hepa1-6 liver cancer bearing (HLCB) mice and its regulatory mechanism on cell apoptosis.
Methods: A total of 40 male C57BL/6 mice were randomly divided into control, moxibustion, cyclophosphamide (CTX) and moxibustion+CTX groups, with 10 mice in each group. The HLCB model was established by subcutaneous inoculation of Hepa1-6 cancer cells into the right armpit. Mice of the CTX and moxibustion+CTX groups were given intraperitoneal injection of CTX (30 mg/kg), once daily for 3 days. Moxibustion with seed-sized moxa cones were applied to "Dazhui" (GV14), bilateral"Zusanli" (ST36) and "Sanyinjiao" (SP6), with 5 moxa cones for each acupoint, once daily for 10 consecutive days. The survival status scores and body weight of HLCB mice were observed, and the tumor weight and tumor inhibition rate were detected. HE staining was used to observe the morphological changes of tumor tissue. ELISA was used to detect the levels of serum interleukin (IL)-2, IL-4 and tumor necrosis factor-α (TNF-α). Western blot and fluorescent quantitative real-time PCR were used to detect the protein and mRNA expressions of cysteine aspartate protease (Caspase) -3 and Caspase-9 in tumor tissues, separately.
Results: Compared with the control group, the survival status scores, body weight, serum IL-2 and TNF-α levels were significantly increased (P<0.05, P<0.01), the tumor weight and serum IL-4 levels were significantly decreased (P<0.05) in the moxibustion group；while the survival status, body weight, tumor weight, serum IL-2 and IL-4 levels were significantly decreased (P<0.01, P<0.05), the content of TNF-α was significantly increased (P<0.01) in the CTX group. The protein and mRNA expressions of Caspase-3 and Caspase-9 in the 3 trentment groups were significantly increased (P<0.05, P<0.01). In comparison with the moxibustion group, the survival status scores, body weight and tumor weight, serum content of IL-2 were significantly decreased (P<0.01, P<0.05). In contrast to the CTX group, the survival status scores, body weight, serum IL-2 and TNF-α content, and the expressions of Caspase-3 and Caspase-9 in tumor tissue were significantly increased (P<0.01, P<0.05), and the tumor weight and serum IL-4 content were significantly decreased (P<0.05) in the moxibustion+CTX group. Results of HE staining showed that the tumor cells in the control group had clear nuclear membranes and nucleoli, with more dividing cells； while less nuclear division and an increase in tumor necrosis areas were found in the 3 treatment groups.
Conclusion: Moxibustion with seed-size moxa cone can enhance the anti-tumor effect of CTX and improve the quality of life of HLCB mice, which may be related with its effect in activating the expressions of Caspase-3 and Caspase-9 in tumor tissue.
目的：探讨麦粒灸对Hepa1-6肝癌荷瘤小鼠的抗肿瘤作用及对细胞凋亡的调控机制。方法：将C57BL/6雄性小鼠随机分为对照组、麦粒灸组、环磷酰胺（CTX）组、麦粒灸＋CTX组，每组10只。将Hepa1-6肿瘤细胞接种于小鼠右侧腋后皮下建立Hepa1-6肝癌荷瘤小鼠模型。CTX组、麦粒灸＋CTX组小鼠给予腹腔注射CTX 30 mg/kg，1次/d，连续3 d；麦粒灸组、麦粒灸＋CTX组小鼠给予麦粒灸“大椎”及双侧“足三里”“三阴交”，每穴5壮，1次/d，连续10 d。观察荷瘤小鼠的生存状态评分和体质量变化，测量瘤体质量，计算抑瘤率；HE染色法观察肿瘤组织病理形态学变化；ELISA法检测血清白细胞介素（IL）-2、IL-4、肿瘤坏死因子-α（TNF-α）的含量；Western blot法和实时荧光定量PCR法检测肿瘤组织中半胱氨酸天冬氨酸蛋白酶（Caspase）-3、Caspase-9蛋白及mRNA的表达。结果：与对照组比较，治疗结束后麦粒灸组小鼠的生存状态评分、体质量、IL-2、TNF-α含量显著升高（P<0.05，P<0.01），瘤体质量、血清IL-4含量显著下降（P<0.05）；CTX组小鼠生存状态评分、体质量及瘤体质量、血清IL-2、IL-4含量显著下降（P<0.01，P<0.05），TNF-α含量显著升高（P<0.01）；3个治疗组肿瘤组织中Caspase-3、Caspase-9蛋白及mRNA表达显著升高（P<0.05，P<0.01）。与麦粒灸组比较，CTX组生存状态评分显著下降（P<0.01），治疗结束后小鼠体质量、瘤体质量、血清IL-2含量显著下降（P<0.01，P<0.05）；与CTX组比较，治疗结束后麦粒灸+CTX组小鼠生存状态评分、体质量、血清IL-2、TNF-α含量、肿瘤组织中Caspase-3、Caspase-9的蛋白和mRNA表达显著升高（P<0.01，P<0.05），瘤体质量、血清IL-4含量显著下降（P<0.05）。对照组肿瘤细胞核膜核仁清晰，分裂细胞多；麦粒灸组肿瘤细胞细胞核分裂较少，肿瘤坏死区较对照组增多；CTX组肿瘤细胞结构较模糊，坏死区较麦粒灸组增多；麦粒灸＋CTX组肿瘤细胞排列松散，核分裂减少，坏死区较CTX组明显增多。结论：麦粒灸能够增强CTX的抗肿瘤作用，提高荷瘤小鼠生存质量，其机制可能与激活肿瘤组织中凋亡相关蛋白Caspase-3、Caspase-9的表达有关。.
Keywords: Caspase-3; Caspase-9; Cyclophosphamide; Liver cancer bearing mice; Moxibustion with seed-size moxa cone.