Objective: To investigate the effect and mechanism of acupoint injection with 0.1% vitamin C+vitamin B complex solution (VC+VBCo) at "Tiantu" (CV 22), "Quchi" (LI 11) and "Zusanli" (ST 36) in mouse model of pneumonia induced by influenza A virus (A/PR/8/34 [H1N1], PR8).
Methods: Sixty male ICR mice were randomized into 6 groups, i.e. control group, model group, acupoint injection group, intraperitoneal injection group, non-target point group and ribavirin group, 10 mice in each one. Except the control group, the pneumonia models were induced by slow nasal dripping PR8 virus in the other groups. On the 2nd day of experiment, VC+VBCo solution, 40 μL was injected at "Tiantu" (CV 22), "Quchi" (LI 11, left) and "Zusanli" (ST 36, left) in the acupoint injection group; VC+VBCo solution, 120 μL was injected intraperitoneally in the intraperitoneal injection group; VC+VBCo solution, 40 μL was injected at non-target acupoints (0.5 cm away from "Tiantu" [CV 22] to the left side, "Quchi" [LI 11, left] and "Zusanli" [ST 36, left]) in the non-target point group; and ribavirin solution, 120 μL was injected intraperitoneally in the ribavirin group. The intervention was delivered once daily, for consecutive 7 days. Three parallel experiments were undertaken. The mean death rate and survival time were assessed in each group, the body mass and lung index were compared among groups. Using HE staining, the morphology of lung tissue was observed; and with real-time fluorescence quantitative PCR, viral load in lung tissue was detected. The concentrations of inflammatory factors (tumor necrosis factor α [TNF-α], interleukin [IL]-1β, IL-10) were detected in lung tissue of each group using ELISA; and those of oxidative stress markers (superoxide dismutase [SOD], glutathione peroxidase [GSH-Px], malondialdehyde [MDA]) were detected with chemiluminescence method.
Results: Compared with the control group, the body mass was decreased and lung index was increased in the model group (P<0.01). In comparison with the model group, body mass was increased in the acupoint injection group (P<0.05), lung index was reduced in the acupoint injection group the and ribavirin group (P<0.05); the mean death rate was decreased and the mean survival time prolonged in the mice of the acupoint injection group (P<0.01, P<0.05); and the mean death rate was reduced in the mice of the ribavirin group (P<0.05). In the model group, the alveolar structure was not integral, the alveolar septum was thickened, inflammatory cells were infiltrated and red blood cells exudated seriously (P<0.01). Compared with the model group, in the acupoint injection group and the ribavirin group, the alveolar structure was integral, the thickened alveolar septum was alleviated; and the infiltration of inflammatory cells and the exudation of red blood cells were reduced remarkably. The viral load was reduced in the mice of the ribavirin group when compared with the model group (P<0.01). Compared with the control group, the concentrations of TNF-α, IL-1β and MDA in lung tissue were increased and those of IL-10, SOD and GSH-Px were reduced in the model group (P<0.01). In the acupoint injection group and the ribavirin group, the concentrations of TNF-α, IL-1β and MDA were reduced in lung tissue and those of IL-10, SOD and GSH-Px were increased (P<0.05, P<0.01) when compared with the model group.
Conclusion: Acupoint injection with VC+VBCo solution may alleviate inflammatory responses and oxidative stress in lung tissue of the PR8-induced pneumonia mice, improve survival rate and prolong the survival time in the case of no effect of the viral load.
目的：探讨“天突”“曲池”“足三里”注射0.1%维生素C（VC）+复合维生素B（VBCo）溶液（VC+VBCo）对甲型流感病毒A/PR/8/34株[influenza A virus A/PR/8/34 （H1N1），PR8]诱导小鼠肺炎模型的作用及机制。方法：将60只雄性ICR小鼠随机分为空白组、模型组、穴位注射组、腹腔注射组、非穴组、利巴韦林组，每组10只。除空白组外，其他各组小鼠经鼻孔缓慢滴入50 μL PR8病毒液诱导肺炎模型。实验第2天，穴位注射组小鼠分别在“天突”“曲池”（左侧）、“足三里”（左侧）注射40 μL的VC+VBCo溶液；腹腔注射组小鼠腹腔注射120 μL的VC+VBCo溶液；非穴组小鼠分别在“天突”左侧和左侧“曲池”“足三里”外侧平行旁开0.5 cm处注射40 μL的VC+VBCo溶液；利巴韦林组小鼠腹腔注射120 μL利巴韦林溶液，均每天1次，连续7 d。进行3次平行实验。统计各组小鼠平均死亡率、存活时间，比较各组小鼠体质量、肺指数，HE染色法观察各组小鼠肺组织形态，实时荧光定量PCR法检测各组小鼠肺组织病毒载量，ELISA法检测各组小鼠肺组织炎性因子[肿瘤坏死因子α（TNF-α）、白介素（IL）-1β、IL-10]含量，化学发光法检测各组小鼠肺组织氧化应激指标[超氧化物歧化酶（SOD）、谷胱 甘肽过氧化物酶（GSH-Px）、丙二醛（MDA）]含量。结果：与空白组比较，模型组小鼠体质量降低、肺指数升高 （P<0.01）；与模型组比较，穴位注射组小鼠体质量升高（P<0.05），穴位注射组、利巴韦林组小鼠肺指数降低（P<0.05）。穴位注射组小鼠平均死亡率降低、平均存活时间延长（P<0.01，P<0.05），利巴韦林组小鼠平均死亡率降低（P<0.05）。模型组小鼠肺泡结构不完整，肺泡间隔增厚，炎性细胞浸润、红细胞渗出严重；与模型组比较，穴位注射组、利巴韦林组小鼠肺泡结构较完整，肺泡间隔增厚减轻，炎性细胞浸润、红细胞渗出明显减少。与模型组比较，利巴韦林组小鼠肺组织病毒载量降低（P<0.01）。与空白组比较，模型组小鼠肺组织TNF-α、IL-1β、MDA含量升高（P<0.01），IL-10、SOD、GSH-Px含量降低（P<0.01）；与模型组比较，穴位注射组、利巴韦林组小鼠肺组织TNF-α、IL-1β、MDA含量降低（P<0.05，P<0.01），IL-10、SOD、GSH-Px含量升高（P<0.01，P<0.05）。结论：VC+VBCo穴位注射可在对病毒载量无影响的情况下，减轻PR8诱导的肺炎小鼠肺组织炎性反应和氧化应激，提高存活率、延长存活时间。.
Keywords: acupoint injection; inflammatory response; influenza A virus; oxidative stress; viral pneumonia.