穿心莲内酯恢复突变型p53野生型功能的作用及机制研究

目的 p53是人体内重要的肿瘤抑制因子,但在人类肿瘤中因高频突变而失去抑癌功能。突变型p53（mutant p53,mutp53）可促进肿瘤的发生、发展和转移。由于在肿瘤细胞中通常有较高表达,mutp53已成为区别于正常细胞的一个特异性抗肿瘤靶点。本研究旨在探索穿心莲内酯的抗肿瘤作用机制,为寻找靶向mutp53的抗肿瘤化合物提供理论依据。方法 构建可以快速筛选具有恢复mutp53下游转录因子的荧光素酶系统,观察穿心莲内酯对H1299-p53 R273H-PUMA-luciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达情况;采用免疫荧光实验,检测穿心莲内酯对HT29（R273H）和SK-BR-3（R175H）细胞中mutp53的表达影响;采用免疫印迹实验进一步观察穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达;随后采用MTT和流式细胞分析,检测穿心莲内酯对肿瘤细胞增殖和凋亡的影响;此外,还通过siRNA敲低Hsp70表达后,研究穿心莲内酯对mutp53下游基因的重激活作用。结果 穿心莲内酯可以增加H1299-p53 R273H-PUMA-luciferase和H1299-p53R175H-PUMA-luciferase细胞中PUMA基因的表达;穿心莲内酯可以降低HT29（R273H）和SK-BR-3（R175H）细胞中mutp53的比例,同时增加野生型p53的比例;穿心莲内酯恢复了mutp53肿瘤细胞中p53下游靶蛋白PUMA、p21、Noxa的表达,进而抑制肿瘤细胞增殖和诱导凋亡;穿心莲内酯可以增加分子伴侣Hsp70的表达,通过siRNA敲低Hsp70后,穿心莲内酯对mutp53下游基因的重激活作用明显受到抑制。结论 穿心莲内酯可能通过影响Hsp70的表达从而激活突变p53下游靶基因而发挥抗肿瘤作用。     

Andrographolide prevents human nucleus pulposus cells against degeneration by inhibiting the NF-κB pathway

Intervertebral disc degeneration (IDD) is among the most common spinal disorders, pathologically characterized by excessive cell apoptosis and production of proinflammatory factors. Pharmacological targeting of nucleus pulposus (NP) degeneration may hold promise in IDD therapy, but it is limited by adverse side effects and nonspecificity of drugs. In this study, we used a natural compound, andrographolide (ANDRO), which has been widely used to intervene inflammatory and apoptotic diseases in the investigation of NP degeneration based on IDD-patients-derived NP cells by lipopolysaccharide (LPS) treatment for the preservation of degeneration. The results showed that LPS maintained the degeneration status of NP cells as evidenced by a high apoptosis rate and the expression of degenerative and inflammatory mediators after LPS treatment. ANDRO reversed the effects of LPS-caused degeneration of NP cells and maintained the phenotype of NP cells, as demonstrated by flow cytometry, degenerative mediators (ADAMTS4 and ADAMTS5), inflammatory factors (COX2, PGE2, MMP-13, and MMP-3), biomarkers of NP cells (SOX9, ACAN, and COL2A1) expressions, and glycosaminoglycan secretion. We also found the involvement of the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) pathway in ANDRO treatment, indicating that ANDRO prevented the LPS-preserved degeneration of NP cells by inhibiting the NF-κB pathway. This study may provide a reference for clinic medication of IDD therapy.     

Synthesis,structure–activity relationships and biological evaluation of dehydroandrographolide and andrographolide derivatives as novel anti-hepatitis B virus agents

Dehydroandrographolide and andrographolide, two natural diterpenoids isolated from Andrographis paniculata possessed activity against HBV DNA replication with IC₅₀ values of 22.58 and 54.07 μM and low SI values of 8.7 and 3.7 in our random assay. Consequently, 48 derivatives of dehydroandrographolide and andrographolide were synthesized and evaluated for their anti-HBV properties to yield a series of active derivatives with lower cytotoxicity, including 14 derivatives against HBsAg secretion, 19 derivatives against HBeAg secretion and 38 derivatives against HBV DNA replication. Interestingly, compound 4e could inhibit not only HBsAg and HBeAg secretions but also HBV DNA replication with SI values of 20.3, 125.0 and 104.9. Furthermore, the most active compound 2c with SI value higher than 165.1 inhibiting HBV DNA replication was revealed with the optimal log P value of 1.78 and log D values. Structure–activity relationships (SARs) of the derivatives were disclosed for guiding the future research toward the discovery of new anti-HBV drugs.     

Physiological Studies on Thiobacilli

Elemental sulfur was metabolized in the colloidal state by cell-free extracts of T. thiooxidans. The activity was found in the soluble fraction (in the supernatant of centrifugation at 130,000 × g for 1 hr). The reaction products were sulfide and thiosulfate. p-Chloromercuribenzoate, acetate monoiodide and potassium cyanide inhibited the reaction.     

Albumin Supplement Affects the Metabolism and Metabolism‐Related Drug–Drug Interaction of Fenoprofen Enantiomers

The influence of albumin towards the metabolism behavior of fenoprofen enantiomers and relevant drug–drug interaction was investigated in the present study. The metabolic behavior of fenoprofen enantiomers was compared in a phase II metabolic incubation system with and without bovine serum albumin (BSA). BSA supplement increased the binding affinity parameter (Km) of (R)‐fenoprofen towards human liver microsomes (HLMs) from 148.3 to 214.4 μM. In contrast, BSA supplement decreased the Km of (S)‐fenoprofen towards HLMs from 218.2 to 123.5 μM. For maximum reaction velocity (Vmax), the addition of BSA increased the Vmax of (R)‐fenoprofen from 1.3 to 1.6 nmol/min/mg protein. In the contrast, BSA supplement decreased the Vmax value from 3.3 to 1.5 nmol/min/mg protein. Andrographolide–fenoprofen interaction was used as an example to investigate the influence of BSA supplement towards fenoprofen‐relevant drug–drug interaction. The addition of 0.2% BSA in the incubation system significantly decreased the inhibition potential of andrographolide towards (R)‐fenoprofen metabolism (P < 0.001). Different from (R)‐fenoprofen, the addition of BSA significantly increased the inhibition potential of andrographolide towards the metabolism of (S)‐fenoprofen. BSA supplement also changed the inhibition kinetic type and parameter of andrographolide towards the metabolism of (S)‐fenoprofen. In conclusion, albumin supplement changes the metabolic behavior of fenoprofen enantiomers and the fenoprofen–andrographolide interaction. Chirality 27:436–440, 2015. © 2015 Wiley Periodicals, Inc.     

玄参的化学成分研究

利用正、反相硅胶以及各种色谱技术进行分离,经理化性质和光谱分析鉴定中药玄参(Scrophularia ningpoensis)有效成分。从玄参(S. ningpoensis)根的乙醇提取物中分离了8个化合物,分别鉴定为14-去氧-12(R)-磺酸基穿心莲内酯（14-deoxy-12(R)-sulfo andrographolide, Ⅷ）、肉桂酸（cinnamic acid,Ⅰ）、对羟基肉桂酸（p-hydroxycinnamic acid,Ⅱ）、丁二酸（succinic acid,Ⅲ）、β-谷甾醇（β-sitosterol, Ⅳ）、胡萝卜甙（daucosteol,Ⅴ）、葡萄糖（gluose,Ⅵ）、熊果酸(ursolic acid,Ⅶ)。Ⅷ为在玄参中首次发现的天然化合物。     

Synthesis and biological evaluation of new C-12(α/β)-(N-) sulfamoyl-phenylamino-14-deoxy-andrographolide derivatives as potent anti-cancer agents

Andrographolide, the major diterpenoidal constituent of Andrographis paniculata (Acanthaceae) and its derivatives have been reported to possess plethora of biological properties including potent anti-cancer activity. In this work, synthesis and in-vitro anti-cancer evaluation of new C-12-substituted aryl amino 14-deoxy-andrographolide derivatives (III a–f) are reported. The substitutions include various sulfonamide moieties –SO₂-NH-R¹. The new derivatives (III a–e) exhibited improved cytotoxicity (GI₅₀, TGI and LC₅₀) compared to andrographolide (I) and the corresponding 3,14,19-O-triacetyl andrographolide (II) when evaluated against 60 NCI cell line panel. Compounds III c and III e are found to be non-toxic to normal human dermal fibroblasts (NHDF) cells compared to reference drug THZ-1.