肠道类器官的实验研究进展

类器官是由多能干细胞或成体干细胞衍生而来的三维组织结构,具有多细胞性和功能性,可以在体外再现活体器官或组织的结构和功能,借以探测干细胞潜力,被誉为“临床前模型”。肠道类器官是将分离后的肠道隐窝置于基质胶中,培养成具有肠道上皮样结构的微型空心球体。肠道类器官的构建,为肠干细胞研究提供了新的手段,使研究者对器官生长成熟和疾病发生的机制有了更深入的了解,为药物筛选提供更为科学的方法和有力证据。

人参皂苷Rg1对辐射致肠IEC-6细胞损伤保护作用的体外实验

目的研究中药单体人参皂苷Rg1对辐射后EC-6细胞的影响及在PI3K/AKT通路的作用机制。方法取大鼠小肠隐窝上皮细胞IEC-6传代培养,设空白组、模型组、中药(人参皂苷)组及中药+抑制剂(LY294002)组,除空白组外,其余各组采用60Co-γ射线照射细胞,剂量为4 Gy。采用CCK8法检测照射后12 h、24 h、36 h时各组细胞增殖活力,流式细胞术检测细胞生存、凋亡情况,Western blot法检测细胞磷酸化蛋白激酶B(phosphorylated AKT,p-AKT)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)及Bcl-2相关X蛋白(Bcl-2 Associated X protein,Bax)的表达,并比较Bax/Bcl-2比值差异。结果CCK-8结果表明,照射后12 h、24 h模型组及中药+抑制剂组增殖活力低于空白组和中药组(P<0.05)。凋亡检测结果显示,照射后各照射组细胞均出现存活率下降、凋亡率升高的趋势;模型组、中药+抑制剂组细胞在12 h、24 h存活率均低于空白组和中药组(P<0.05);至36 h,模型组、中药组与空白组生存率及凋亡率均相似(P>0.05),中药+抑制剂细胞存活率与其他3组相当(P>0.05),但凋亡率显著高于其他3组(P<0.05)。Western blot法检测结果显示,模型组p-AKT、Bcl-2显著低于中药组和空白组(P<0.05),模型组p-AKT显著高于中药+抑制剂组(P<0.05);模型组和中药+抑制剂组Bax及Bax/Bcl-2比值显著高于空白组和中药组(P<0.05)。结论人参皂苷Rg1能够有效促进辐射后肠IEC-6细胞存活、抑制凋亡,其作用机制可能与其促进PI3K/AKT相关因子表达有关。

《中医治未病实践指南》中药茶调理偏颇体质的一致性测评分析

目的:系统分析《中医治未病实践指南》(指南)中所推荐的调理偏颇体质的药茶在实践测评中是否与指南一致。方法:通过全国10家一致性测评单位的实践测评,对推荐的21种药茶进行对应的体质调理,每种偏颇体质对应2~3种药茶,共测评241例,调理时间为1个月,观察受试者对药茶的接受度和口感、服用药茶后症状改善情况和体质疗效指数以及不良反应等指标。结果:受试者对指南中推荐的药茶的接受度和口感,其中口感非常满意率为34.4%,满意率达98.3%左右,偏颇体质倾向症状改善率为100.0%,体质疗效指数中总有效率约76.8%,不良反应率为3.7%,仅1例退出,其他减量后不良反应缓解。结论:指南中所推荐的调理偏颇体质的药茶在实践测评中与指南一致,容易被患者接受且具有较好的口感和较好的调理效果,安全性能好。

响应曲面法优化马齿苋中脂肪酸提取工艺及其成分分析

目的:优选新鲜马齿苋脂肪酸的提取工艺,对新鲜马齿苋全草油进行含量测定、理化指标测定及脂肪酸成分分析。方法:以α-亚麻酸提取率为评价指标,采用高效液相色谱法检测,通过单因素及响应曲面法考察料液比、超声温度、超声时间对脂肪酸提取工艺的影响,并确定最佳工艺参数。参考食品安全国家标准对新鲜马齿苋全草油理化指标进行评价。采用气相色谱-质谱联用法对脂肪酸成分进行分析。结果:以料液比1∶22、超声温度70℃、超声时间61min为最佳提取工艺,此时α-亚麻酸提取率为4.210%,多不饱和脂肪酸相对含量为27.6104%。结论:利用响应曲面法优化新鲜马齿苋脂肪酸提取工艺,并结合色谱法分析其组成的方法可行性较好,操作简单便捷,对药食同源中药材的进一步探究具有重要意义。

基于望诊的急性胃肠道辐射伤大鼠模型中医证候演变规律

目的通过观察实验动物活体、器官和组织动态变化,总结急性胃肠道辐射伤的中医证候学特点及演变规律。方法将90只SD雄性大鼠随机分为模型组80只和对照组10只。模型组采用60Co-γ射线单次全身照射,对照组不予照射,观察大鼠全身状况,并分别于照射后第1、4、7、10、14天肉眼及显微镜观察胃肠道及组织形态变化。结果照射后初期(0~2天),模型组出现躁动、多饮、目赤等现象,小肠黏膜轻度充血水肿;早期(3~6天),体质量下降,水样便,小肠黏膜充血水肿明显、多发出血灶及糜烂坏死;中期(7~12天),经过短暂恢复后体质量继续下降、拒食、大量死亡,胃肠腔内淤积紫黑色黏液,黏膜下层微血管闭塞;后期或恢复初期(13天后),蜷缩少动、毛发干枯、爪甲苍白,肠组织固有层间质纤维增加。对照组活体、胃肠及黏膜组织观察均无异常。结论急性胃肠道辐射损伤,初期属热毒内盛、气血两燔证,早期为热灼血肉、元气耗伤、脾阳不振证,中期为元气虚损、真阴耗伤、瘀毒互结证,后期为肝肾亏耗、气血两虚、血络瘀阻证。

健脾益气类中药减轻含顺铂化疗方案消化道反应的Meta分析

目的分析健脾益气法减轻含顺铂方案化疗患者消化道反应的临床疗效。方法检索Pubmed、Embase、Cochrane Library、Sino Med、中国知网、万方数据、维普网数据库中符合纳入标准的文献,检索时间为建库至2017年4月25日。使用Jadad量表评价文献质量后纳入Meta分析。结果共纳入24个随机对照试验文献,其中中西医联合组患者848例,单纯化疗组816例。纳入文献质量为1~3分。Meta分析结果显示,中西医联合组在恶心呕吐[RR=0.71,95%CI(0.64,0.78),P<0.001]、食欲不振[RR=0.51,95%CI(0.34,0.76),P<0.001]、排便异常[RR=0.62,95%CI(0.45,0.86),P=0.004]、肿瘤疗效指标[RR=1.14,95%CI(1.07,1.21),P<0.001]和生活质量[RR=1.59,95%CI(1.38,1.83),P<0.001]方面,中西医联合组亦优于单纯化疗组。结论健脾益气类中药可减少化疗患者恶心呕吐、食欲不振、排便异常发生率,而且对化疗效果和生活质量也有改善作用。

Extracts from Huangqi(Radix Astragali Mongoliciplus) and Ezhu(Rhizoma Curcumae Phaeocaulis) inhibit Lewis lung carcinoma cell growth in a xenograft mouse model by impairing mitogen-activated protein kinase signaling, vascular endothelial growth factor prod

OBJECTIVE: To study the anti-tumor effects of the extracts from Huangqi(Radix Astragali Mongolici)and Ezhu(Rhizoma Curcumae Phaeocaulis) on the growth of Lewis lung carcinoma(LLC) in a xenograft mouse model and to investigate the possible underlying mechanism.METHODS: LLC tumor-bearing C57 BL/6 mice were treated with normal saline, cisplatin(2 mg/kg intraperitoneally every other day), or Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis)(1∶1, 2∶1, or 3∶1 ratio;5, 8, or 11 g/kg crude drug intragastrically every day) for 15 d.Body weights and tumor volumes were measured every other day. Tumors were excised on day 15 and analyzed. Tumor microvessel density(MVD)was assessed by immunohistochemical staining of CD34;and expression of vascular endothelial cell growth factor(VEGF), the mitogen-activated protein kinases p38 mitogen-activated protein kinase(MAPK), extracellular signal-regulated kinases 1 and 2(ERK1/2), and Jun N-terminal kinase(JNK)and their phosphorylated forms were assessed by Western blotting.RESULTS: Treatment with cisplatin caused a significant loss of body weight compared with controls,whereas Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) extract combinations had no effect. Extracts from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) significantly decreased tumor weight and tumor MVD compared with controls,and at the 3∶1 treatment group had similar efficacy to cisplatin in reducing MVD. Tumors from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) treatments also showed decreased p38 MAPK, p-p38 MAPK, ERK1/2, p-ERK1/2,JNK, and p-JNK expression compared with the control group(all P < 0.01). VEGF protein expression was significantly reduced in the 2∶1 and 3∶1 treatment groups compared with the control group(P < 0.01).CONCLUSION: Extracts from Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) hindered LLC growth in the xenograft mouse model, possibly via inhibition of the MAPK signaling pathway, VEGF production, and tumor angiogenesis.

Bupi Hewei decoction ameliorates 5-fluorouracil-induced intestinal dysbiosis in rats through T helper 17/T regulatory cell signaling pathway

OBJECTIVE:To observe the effects of the Bupi Hewei(BPHW)decoction on diarrhea and intestinal flora disorder induced by 5-fluorouracil(5-FU)and investigate the possible mechanism underlying these actions.METHODS:Thirty-five male Sprague-Dawley rats were randomly divided into four groups:normal control,5-FU,5-FU+BPHW decoction(10.5 g/kg for 5 consecutive days),and 5-FU+Bacillus licheni-formis capsule groups(0.2 g/kg for 5 consecutive days).Animal models were established via the intraperitoneal injection of 5-FU(30 mg/kg for 5 consecutive days).At the end of the treatment period,diarrhea was assessed,and the change of the intestinal flora was examined using 16 S r DNA highthroughput sequencing.Interleukin(IL)-17,IL-21,IL-6,IL-10,RAR-related orphan receptor gamma(RORγt),and forkhead box P3(Foxp3)expression in the jejunum was detected using immunohistochemistry,quantitative real-time polymerase chain reaction(PCR),Western blotting,and enzymelinked immuno sorbent assay.RESULTS:In this study,the BPHW decoction effectively lowered the diarrhea score,increased the proportions of Bacteroidetes and Prevotellaceae-Alloprevotella species,and reduced the proportions of Proteobacteria,Escherichia-Shigella,Ruminococcaceae NK4 A214,and Ruminococcaceae UCG-005 species in the rat intestine after 5-FU chemotherapy.In addition,the BPHW decoction significantly suppressed the expression of IL-17,IL-21,IL-6,IL-10,RORγt,and Foxp3 in the jejunum.CONCLUSION:Our findings suggest that the BPHW decoction can improve the intestinal immune balance and reduce intestinal inflammation by targeting T helper cell/T regulatory cell-associated factors.

Effect of optimal combination of Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) on proliferation and apoptosis of A549 lung cancer cells

OBJECTIVE: To investigate the effect of optimal combination(E) of Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) on proliferation and apoptosis of A549 lung cancer cells and the possible mechanism underpinning the action.METHODS: A uniform design method was used to optimize the E of Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) in A549 lung cancer cells. MTS assay was applied to analyze the effect of the component formula ofHuangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Curcumae Phaeocaulis) on A549 cells viability in various uniform design groups. A549 cells with exponential growth in routine culture were exposed to Co Cl_2(200 μmol/L) to mimic hypoxic conditions. Group 0 was treated with RPMI-1640, the group Co Cl_2 was treated with Co Cl_2(200 μmol/L),the group DDP + Co Cl_2 was treated with 4 mg/L Cisplatin injection(DDP) + Co Cl_2(200 μmol/L), and the drug group was treated with various dose of E(0.5 E, 1 E, 2 E) + Co Cl_2(200 μmol/L). All groups were cultured for 24 h. Cell apoptosis was measured by Annexin V-FITC/propidium iodide double staining and flow cytometry. Western blot assay and quantitative real-time polymerase chain reaction(q RT-PCR) were employed to detect the protein and m RNA expression of B-celllymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax) and cysteinyl aspartate specific proteinase-3(caspase-3).RESULTS: The E obtained by the uniform design was comprise of 200 mg/L Astragalus polysaccharide(X1) and 32 mg/L Curcumin(X3). Group DDP+Co Cl_2, group 1 E + Co Cl_2 and group 2 E + Co Cl_2 promoted the apoptosis of A549 cells(P < 0.05). Group1 E + Co Cl_2 and group 2 E + Co Cl_2 had no statistically significant differences compared with the group DDP + Co Cl_2(P > 0.05). Compared with group 0, various doses of E + Co Cl_2 could up-regulate the expression of Bax and caspase-3 and down-regulate the expression of Bcl-2 at protein and m RNA levels(P < 0.05).CONCLUSION: Astragalus polysaccharide and Curcumin was the optimal combination of Huangqi(Radix Astragali Mongolici) and Ezhu(Rhizoma Cur-cumae Phaeocaulis). E promoted the apoptosis of A549 cells. Combination of Astragalus polysaccharide and Curcumin increased the expression of Bax and caspase-3, and decreased the expression of Bcl-2 to initiate apoptosis in A549 cells under chemical-induced hypoxia.

Efficacy of Liangxue Guyuan decoction(凉血固元汤)on radiation-induced intestinal injury in rats via the toll-like receptor 4/myeloid differentiation primary response 88/nuclear factor-kappa B pathway

OBJECTIVE:To evaluate the efficacy of Liangxue Guyuan decoction(凉血固元汤,LGD)on radiation-induced intestinal injury in rats,and the possible underlying mechanism of action.METHODS:A total of 255 male Sprague-Dawley rats were used.15 rats were assigned to the control group and the remaining 240 rats were exposed to a^(60)Co source at a dose of 11 Gy.Irradiated rats were randomly divided into model,dexamethasone(DXM),low-dose LGD(LGDl),and high-dose LGD(LGDh)groups and treated for 11 d.The survival rate,weight of body,intestinal pathology and the expression of toll-like receptor 4(TLR4),myeloid differentiation primary response 88(MyD88),and nuclear`factor-kappa B(NF-κB)were recorded.RESULTS:Radiation reduced the survival rate and weight of rats,destroyed the intestinal structure,induced an inflammatory reaction,and increased both protein and mRNA expression of TLR4,MyD88,and NF-κB in ileum.However,LGDh increased the survival rate,inhibited weight loss,alleviated inflammation and improve the expression of TLR4 pathway.CONCLUSIONS:LGD increased the survival rate and inhibit weight loss of irradiated rats,and reduced inflammation and intestinal injury.The underlying mechanism may involve regulation of the TLR4/MyD88/NF-κB pathway.