人参皂苷Rg3治疗放射性直肠炎大鼠的作用机制研究

Investigation of the mechanism of ginsenoside Rg3 in the treatment of radiation proctitis in rats＊

摘要:

目的 基于TNF-α/NF-κB及Caspase-8信号通路，研究人参皂苷Rg3（GRg3）治疗放射性直肠炎大鼠的机制。方法 48只雄性Wistar大鼠随机分为对照组、模型组、地塞米松组、低剂量GRg3治疗组、中剂量GRg3治疗组、高剂量GRg3治疗组，每组8只。6MV X射线单次21.5 Gy腹部照射进行模型复制，照射后第8天开始给药，观察大鼠情况并称重，2周后腹主动脉取血并处死解剖。HE染色观察直肠组织病理学变化，酶联免疫吸附试验检测血清TNF-α、IL-4、IL-10水平，实时荧光定量聚合酶链反应（qRT-PCR）检测直肠组织IKK-β、IκB-α、Caspase-8基因表达，Western blotting检测IKK-β、IκB-α、p-IκB-α、NF-κB p50、Caspase-8蛋白表达。结果 照射后第6天，与对照组比较，其他各组大鼠体重明显降低（P <0.05），且除对照组外的其他组大鼠体重比较，差异无统计学意义（P >0.05）；开始灌胃后，对照组大鼠体重逐步上升，模型组大鼠体重不断下降，地塞米松组和GRg3治疗组大鼠在治疗前3天体重较治疗前无明显变化，从第4天开始逐渐增长；与模型组相比，灌胃第8、14天，地塞米松组和高剂量GRg3治疗组大鼠体重明显升高（P <0.05）。治疗2周后，地塞米松组直肠组织结构完整，黏膜层见少量的炎症细胞浸润；低剂量GRg3治疗组直肠组织大范围溃疡，肠腺形状、大小不一，数量减少，并伴大量炎症细胞浸润，少量肠腺扩张，腺腔内可见坏死细胞碎片，损伤侵及黏膜下层，黏膜肌层及黏膜下层亦可见炎症细胞浸润；中剂量GRg3治疗组见少量黏膜上皮细胞坏死脱落，肠腺形状、大小不一，伴中等量炎症细胞浸润，少量肠腺扩张，腺腔内可见坏死细胞碎片；高剂量GRg3治疗组直肠组织结构完整，见黏膜层水肿，肠腺排列疏松，伴炎症细胞浸润。与对照组比较，模型组大鼠血清TNF-α水平升高（P <0.05），IL-4、IL-10水平降低（P <0.05）；与模型组比较，地塞米松组及高剂量GRg3治疗组血清TNF-α水平降低（P <0.05），IL-4、IL-10水平升高（P <0.05）。与对照组比较，模型组大鼠直肠组织IKK-β、IκB-α及Caspase-8基因表达升高（P <0.05）；经过不同的干预治疗后，与模型组比较，各治疗组IKK-β、IκB-α及Caspase-8基因表达均降低（P <0.05），且随着GRg3治疗剂量的升高各因子表达呈下降趋势；地塞米松组与高剂量GRg3治疗组IKK-β、IκB-α及Caspase-8 mRNA的表达比较，差异无统计学意义（P >0.05）。与对照组比较，模型组大鼠直肠组织IKK-β、p-IκB-α、胞核NF-κB p50及Caspase-8蛋白表达升高（P <0.05），IκB-α、胞浆NF-κB p50蛋白表达降低（P <0.05）；经过不同治疗后，与模型组相比，地塞米松及中、高剂量GRg3治疗组IKK-β、p-IκB-α、胞核NF-κB p50及Caspase-8蛋白表达均降低（P <0.05），IκB-α及胞浆NF-κB p50蛋白表达均升高（P <0.05）。中、高剂量GRg3治疗组与地塞米松组各因子蛋白表达比较，差异无统计学意义（P >0.05）。结论 GRg3通过抑制IKK-β的表达，减少IκB-α的磷酸化，增加胞浆中NF-κB p50的滞留，减轻放射性直肠炎，促进肠道组织损伤修复；GRg3能抑制电离辐射引起的肠道细胞凋亡减轻炎症反应。

Abstract:

Objective To investigate the mechanism of ginsenoside Rg3 (GRg3) in the treatment of radiation proctitis in rats, this study focuses on the TNF-α/NF-κB and Caspase-8 signaling pathways.Methods A total of 48 male Wistar rats were randomly assigned to control group, model group, dexamethasone group, low-dose GRg3 treatment group, medium-dose GRg3 treatment group, and high-dose GRg3 treatment group, with 8 rats in each group. The model was replicated by a single dose of 21.5 Gy abdominal irradiation using 6MV X-rays. Drug administration began on the 8th day after irradiation. The rats' conditions were observed and their weights were measured. Two weeks later, blood was collected from the abdominal aorta before the rats were euthanized for dissection. Hematoxylin and eosin staining was used to observe pathological changes in the rectal tissue. Enzyme-linked immunosorbent assay (ELISA) was performed to measure serum levels of TNF-α, IL-4, and IL-10. Real-time quantitative polymerase chain reaction (qRT-PCR) was conducted to assess IKK-β, IκB-α, and Caspase-8 mRNA expression in rectal tissue. Western blotting was employed to analyze IKK-β, IκB-α, p-IκB-α, NF-kB p50, and Caspase -8 protein expression.Results On the 6th day after irradiation, the body weight of rats in all groups except the control group showed a significant decrease (P < 0.05), with no statistical significance observed in comparison to each other (P >0.05). Following instillation, the body weight of rats in the control group gradually increased, while those in the model group exhibited continuous decline. The body weight of rats in both the dexamethasone and GRg3 treatment groups remained relatively stable for the first 3 days of treatment, followed by gradual increases from day four onwards. Notably, on the 8th and 14th day of gavage, significant increases were observed in the body weight of rats within these two treatment groups compared to that of the model group (P < 0.05). After a two-week treatment period, rectal tissue structure was found to be intact in the dexamethasone group; however, a small amount of inflammatory cell infiltration was noted within its mucosal layer. Conversely, wide-ranging ulceration along with varied intestinal gland morphology and extensive inflammatory cell infiltration were evident in rectal tissues from low-dose GRg3 treated rats. Compared to controls, serum TNF-α levels were elevated while IL-4 and IL-10 levels were decreased within model group rats (P < 0.05). In contrast, both TNF-α levels decreased significantly (P < 0.05) while IL-4 and IL-10 levels increased significantly (P < 0.05) following treatments with dexamethasone or high-dose GRg3 when compared to model group values. In comparison to the control group, there was an increase in mRNA expression levels for IKK‐β, IκB‐α, and Caspase‐8 observed within the rectal tissue samples from the model group (P < 0.05). Following various intervention treatments, the mRNA expression levels for these factors decreased across all treatment groups when compared to the model cohort (P < 0.05) with a consistent downward trend as GRg3 dosage increased. There was no significant difference in the expression of IKK-β, IκB-α and Caspase-8 mRNA between dexamethasone group and high-dose GRg3 group (P > 0.05). Compared to the control group, the protein expressions of IKK-β, p-iκb-α, nuclear NF-κB p50, and Caspase-8 in the rectal tissue of the model group were found to be increased (P < 0.05), while the protein expressions of IκB-α and cytoplasmic NF-κB p50 were decreased (P < 0.05). Following different treatments, it was observed that compared to the model group, the protein expressions of IKK-β, p-iκb-α, nuclear NF-κB p50, and Caspase-8 in both dexamethasone and medium and high dose GRg3 treatment groups were decreased (P < 0.05), whereas the protein expressions of IκB-α and cytoplasmic NF-κB p50 were increased (P < 0.05). There was no significant difference in protein expression between the dexamethasone group and medium and high dose GRg3 group (P > 0.05).Conclusions GRg3 has been shown to inhibit the expression of IKK-β, reduce the phosphorylation of IκB-α, increase the retention of NF-κB p50 in the cytoplasm, alleviate radiation enteritis, and promote the repair of intestinal tissue damage. Additionally, GRg3 can inhibit intestinal cell apoptosis induced by ionizing radiation and reduce inflammation.