升麻是我国传统的中草药,现代药理研究表明其具有明显的抗病毒活性。许多多糖已被证明具有免疫增强和抗病毒活性,但对升麻多糖(CRP)生物活性的研究较少。目的探讨CRP的特性及其对提高免疫活性和抑制传染性胃肠炎病毒(TGEV)的作用。单糖组成,分子量,傅里叶变换红外光谱和电镜分析测定CRP。用比色法研究CRP对淋巴细胞和RAW264.7细胞免疫活性的影响,FITC-OVA荧光染色和ELISA。使用Real-timePCR测定CRP对TGEV感染的PK-15细胞的影响,Hoechst荧光染色,锥虫蓝染色,吖啶橙染色,膜联蛋白V-FITC/PI荧光染色,DCFH-DA加载探头,和JC-1染色。网络药理学用于预测CRP在增强免疫力和抗TGEV中的作用靶点,和分子对接用于进一步分析CPR和核心靶标之间的结合模式。结果显示,CRP主要由葡萄糖和半乳糖组成,分子量为64.28kDa。CRP组的iNOS和NO含量明显高于对比组。CRP(125和62.5μg/mL)能显著增强RAW264.7细胞的吞噬能力,IL-1β含量与对照组比较。250μg/mLCRP对TGEV有明显的抑制作用,与TGVE组相比,可以显着减少细胞凋亡,与TGVE组相比可以抑制线粒体膜电位的降低。CRP组TGEVN基因mRNA表达显著低于TGEV组。PPI显示免疫增强的核心靶点是AKT1、MMP9、HSP90AA1等。,TGE的核心靶点是CASP3、MMP9、EGFR、等。分子对接表明CRP具有与靶标结合的潜力。这些结果表明CRP具有更好的免疫增强作用和抗TGEV活性。
Cimicifugae rhizoma is a traditional Chinese herbal medicine in China, and modern pharmacological research showed that it has obvious antiviral activity. Many polysaccharides have been proved to have immune enhancement and antiviral activity, but there are few studies on the biological activity of Cimicifuga rhizoma polysaccharide (CRP). The aim was to explore the character of CRP and its effects on improving immune activity and inhibiting transmissible gastroenteritis virus (TGEV). The monosaccharide composition, molecular weight, fourier transform infrared spectra and electron microscopy analysis of CRP was measured. The effect of CRP on immune activity in lymphocytes and RAW264.7 cells were studied by colorimetry, FITC-OVA fluorescent staining and ELISA. The effect of CRP on TGEV-infected PK-15 cells was determined using Real-time PCR, Hoechst fluorescence staining, trypan blue staining, acridine orange staining, Annexin V-FITC/PI fluorescent staining, DCFH-DA loading probe, and JC-1 staining. Network pharmacology was used to predict the targets of CRP in enhancing immunity and anti-TGEV, and molecular docking was used to further analyze the binding mode between CPR and core targets. The results showed that CRP was mainly composed of glucose and galactose, and its molecular weight was 64.28 kDa. The content of iNOS and NO in CRP group were significantly higher than the control group. CRP (125 and 62.5 μg/mL) could significantly enhance the phagocytic capacity of RAW264.7 cells, and imprive the content of IL-1β content compared with control group. 250 μg/mL of CRP possessed the significant inhibitory effect on TGEV, which could significantly reduce the apoptosis compared to TGVE group and inhibit the decrease in mitochondrial membrane potential compared to TGVE group. The mRNA expression of TGEV N gene in CRP groups was significantly lower than TGEV group. PPI showed that the core targets of immune-enhancing were AKT1, MMP9, HSP90AA1, etc., and the core targets of TGE were CASP3, MMP9, EGFR, etc. Molecular docking show that CRP has binding potential with target. These results indicated that CRP possessed the better immune enhancement effect and anti-TGEV activity.