羌活中的香豆素类成分及其抑制脂多糖诱导的RAW 264.7细胞NO生成活性的研究

目的研究羌活Notopterygium incisum的香豆素类成分及其抗炎活性。方法采用硅胶、HPLC等柱色谱方法进行分离纯化,通过质谱、核磁共振波谱数据鉴定化合物的结构;采用脂多糖(LPS)诱导的小鼠巨噬细胞RAW 264.7炎症反应模型,考察羌活中香豆素类成分对炎症反应模型一氧化氮(NO)生成的影响。结果从羌活甲醇提取物分离得到24个香豆素类化合物,分别鉴定为异欧前胡素(1)、川白芷素(2)、补骨脂素(3)、香柑内酯(4)、茵陈素(5)、欧芹酚(6)、5-去氢羌活醇(7)、环氧脱水羌活醇(8)、7″-O-甲基异羌活醇(9)、佛手柑素(10)、7-异戊烯氧基-6-甲氧基-香豆素(11)、栓翅芹烯醇(12)、羌活醇(13)、去甲呋喃羽叶芸香素(14)、异羌活醇(15)、蛇床夫内酯(16)、6-异戊烯氧基伞形花内酯(17)、紫花前胡苷元(18)、异虎耳草素(19)、紫花前胡苷(20)、前胡苷V(21)、前胡苷I(22)、印枳苷元-11-O-β-D-吡喃葡萄糖基-(1→6)-β-D-吡喃葡萄糖苷(23)、羌活苷(24)。化合物7～10、13和15抑制LPS诱导的RAW 264.7细胞NO生成活性最强,最大半数抑制浓度(IC_(50))值为8.50～35.12μmol/L。结论化合物7为新的天然产物,化合物17为首次从羌活中分离得到;C-5位上具有多烯烃结构的香豆素抑制LPS诱导的RAW 264.7细胞NO生成活性较强。     

A perivascular niche in the bone marrow hosts quiescent and proliferating tumorigenic colorectal cancer cells

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones—a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.     

Peripheral whole blood FOXP3 TSDR methylation: a potential marker in severity assessment of autoimmune diseases and chronic infections

Immune dysregulation is a cardinal feature of autoimmune diseases and chronic microbial infections. In particular, regulatory T cells are downregulated in autoimmune diseases while upregulated in chronic microbial infections. FOXP3 is the master regulator of Treg development. Treg-specific demethylated region (TSDR) is a highly conserved locus on the FOXP3 gene that is fully demethylated in natural Tregs but methylated in effector T cells. In our study, we used high resolution melt-polymerase chain reaction (HRM-PCR) to determine the FOXP3 TSDR methylation status in autoimmune diseases and chronic microbial infections. We found that FOXP3 TSDR to have the highest mean melting temperature (highly methylated) in active SLE patients compared to all the other groups (p?<?0.001). The psoriasis group also had a significantly high mean melting temperature (78.62?±?0.20) when compared with the inactive SLE group (78.49?±?0.29, p?<?0.05) and control group (78.44?±?0.25, p?<?0.01). There was no significant difference in melting temperature between inactive SLE and healthy controls. Disease activity in SLE was directly associated with methylation of the FOXP3 TSDR. On the other hand, patients with chronic microbial infections had significantly lower FOXP3 TSDR mean melting temperature (demethylated) when compared with healthy controls (78.28?±?0.21 vs 78.44?±?0.25, p?<?0.05). Our results suggest that the use of HRM-PCR to detect FOXP3 TSDR methylation status is a reliable and easy method to predict natural regulatory T cell levels in peripheral blood in different disease conditions. Determining FOXP3 TSDR methylation status can be a useful tool in diagnosis, and monitoring the severity of autoimmune diseases and chronic microbial infections.     

Sirt5 is dispensable for BrafV600E‐mediated cutaneous melanoma development and growth in vivo

Sirt5 is known to functionally regulate mitochondrial proteins by altering posttranslational modifications, including lysine desuccinylation. While roles for Sirt5 as either a tumor promoter or suppressor, or in chemoresistance, have been implicated in other cancers, the function of Sirt5 in cutaneous melanoma has not been well examined. Therefore, to determine whether Sirt5 is necessary for Braf^V600E‐mediated melanoma formation and/or disease progression, we crossed a genetically engineered murine melanoma model (Tyr^CreERT2/+; Braf^{LSL‐V600E/+}; Pten^flox/flox) to Sirt5^?/? knockout animals. In addition, we tested for synergism with a selective BRAF (V600E) inhibitor in Sirt5^?/? mouse melanoma cells. Taken together, this report demonstrates that, in these models, Sirt5 is dispensable for Braf^V600E‐mediated cutaneous melanoma formation and growth in vivo, and does not improve sensitivity to a selective BRAF inhibitor.     

滋阴益肾法治疗特发性血小板减少性紫癜疗效及对患者血清IL-4、IL-6和免疫功能的影响

目的: 探讨滋阴益肾法治疗特发性血小板减少性紫癜(ITP)的疗效及对血清IL-4、IL-6、TNF-α及免疫功能的影响。方法:选取ITP患者84例,随机数字表法分为对照组和观察组,各42例。对照组采用口服泼尼松片治疗,观察组在对照组用药基础上给予滋阴益肾法治疗。比较两组临床疗效,治疗前后IL-4、IL-6、TNF-α、外周血T淋巴细胞亚群(CD₃⁺、CD₄⁺、CD₈⁺、CD₄⁺ /CD₈⁺)及免疫球蛋白水平变化情况。结果:两组治疗后中医症候积分明显降低,观察组优于对照组(P<0.05)。观察组总有效率95.24%高于对照组78.57%(P<0.05); 两组治疗后PLT计数均明显升高、PAIgG表达水平降低,观察组更明显(P<0.05); 两组治疗前IL-4、IL-6、TNF-α及外周血T淋巴细胞亚群水平比较差异无统计学意义(P>0.05); 治疗后CD₃⁺、CD₄⁺、CD₄⁺ /CD₈⁺均升高,IL-4、IL-6、TNF-α、CD₈⁺均降低(P<0.05); 观察组治疗后CD₃⁺、CD₄⁺、CD₄⁺ /CD₈⁺均高于对照组,IL-4、IL-6、TNF-α、CD₈⁺均低于对照组(P<0.05)。治疗后观察组的血清IgA和IgG水平明显高于对照组(P<0.05),而血清IgM水平差异无统计学意义(P>0.05)。两组不良反应情况比较无统计学差异(P>0.05)。结论: 滋阴益肾法可显著降低ITP患者血清IL-4、IL-6等因子水平,提高患者免疫能力。     

红景天苷通过TLR4调控DC提高T细胞对肺癌3LL细胞的杀伤力

目的：探讨红景天苷（salidroside，SAL）对树突状细胞（dendritic cell，DC）表型及细胞毒性T细胞（cytotoxic T lympho‐cyte，CTL）抗肿瘤能力的影响。方法：选用Lewis肺癌细胞株3LL、野生型C57BL/6和TLR4-/-C57BL/6小鼠，获取小鼠骨髓来源的DC前体细胞，经过培养分化成未成熟DC，收获第6天的DC，经磁珠分选后获得纯度较高的CD11c+DC。将细胞分成PBS组、SAL组和脂多糖（lipopolysaccharide，LPS）组，培养48 h后用流式细胞术检测SAL体外对DC表面分子、吞噬功能、TLR4通路和对T细胞杀伤能力的影响。结果：与 PBS 组比较，SAL组DC的表面分子CD80、CD86、MHC Ⅱ表达水平显著升高（均 P<0.05）、吞噬功能显著下降（P<0.05）、TLR4 表达水平显著升高（P<0.01）；与野生型组比较，TLR4-/-组DC经SAL或LPS处理后，其表面分子CD80、CD86、MHC Ⅱ的表达水平显著降低（均P<0.05）；与PBS组比较，SAL组刺激活化的CTL对肺癌3LL细胞的杀伤效应显著升高（P<0.05）。结论：SAL可以通过调控TLR4诱导DC成熟，从而提高T细胞的杀伤能力     

Vasoactive Intestinal Peptide Immunoregulatory Role at the Periapex: Associative and Mechanistic Evidences from Human and Experimental Periapical Lesions

IntroductionThe balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions.MethodsPeriapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects.ResultsVIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4.ConclusionsOur results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells.     

基于子宫内膜异位症与恶性肿瘤的相关性探析补肾化瘀法作用机制及中医理论依据

子宫内膜异位症其异位内膜组织虽然在形态学上呈良性表现，却具有类似恶性肿瘤的生物学特性。中医学一般认为，“正虚伏邪”为恶性肿瘤的病机特点；那么，肾虚血瘀既属于“正虚伏邪”的范畴，又体现了EMs发病学和疾病发生、发展过程中的主要特点。在“病证相应”的中医治则之下，补肾化瘀法的临床疗效主要表现为：缓解痛经症状、提高受孕率，以及调整月经周期。本文基于EMs与恶性肿瘤的相关性，通过分析补肾化瘀法治疗EMs的理论依据及其抑制异位内膜侵袭的调控机制，主要包括解除免疫抑制、阻断局部微血管新生等，旨在阐明其疗效显著的原因，为临床推广提供可靠的基础研究证据。