一氧化氮对重症急性胰腺炎肺损伤的作用及其机制

目的研究一氧化氮(NO)减轻重症急性胰腺炎(SAP)肺损伤的机制。方法采用逆行胰胆管牛磺胆酸钠注射制造SAP大鼠模型。动物分为假手术组、胰腺炎组、L-精氨酸(L-Arg)治疗组和氯喹(CQ)治疗组。硝酸还原酶法检测肺组织NO的浓度变化,实时定量PCR(RT-PCR)检测肺组织TLR(Toll-like receptor)2／4 mRNA表达变化。结果与假手术组比较,SAP大鼠肺组织NO浓度降低,肺损伤加重;肺组织TLR2／4 mRNA表达增高,肺组织肿瘤坏死因子-α(TNF-α)表达升高(P<0．05)。给予不同剂量L-Arg治疗后,肺组织NO浓度明显升高,肺损伤程度减轻;TLR2／4 mRNA表达降低,肺组织TNF-α表达降低(P<0．05)。给予CQ抑制肺组织TLR2／4 mRNA表达后,肺组织内NO浓度升高,肺损伤减轻,TNF-α表达降低(P<0．05)。结论NO可以明显抑制SAP肺组织TLR2／4 mRNA的表达,减少细胞因子的合成及释放,从而减轻肺组织损伤。     

Piperine inhibits the proliferation of human prostate cancer cells via induction of cell cycle arrest and autophagy

Piperine, an alkaloid from black and long peppers (Piper nigrum Linn & Piper longum Linn), has been reported to exhibit antitumor activities in vitro and in vivo. To further understand the antitumor mechanism of piperine, we investigated the growth inhibitory effects of piperine on human prostate cancer DU145, PC-3 and LNCaP cells. Piperine treatment resulted in a dose-dependent inhibition of the proliferation of these cell lines. Cell cycle arrest at G₀/G₁ was induced and cyclin D1 and cyclin A were downregulated upon piperine treatment. Notably, the level of p21^Cip1 and p27^Kip1 was increased dose-dependently by piperine treatment in both LNCaP and DU145 but not in PC-3 cells, in line with more robust cell cycle arrest in the former two cell lines than the latter one. Although piperine induced low levels of apoptosis, it promoted autophagy as evidenced by the increased level of LC3B-II and the formation of LC3B puncta in LNCaP and PC-3 cells. The piperine-induced autophagic flux was further confirmed by assaying LC3-II accumulation and LC3B puncta formation in the presence of chloroquine, a well-known autophagy inhibitor. Taken together, these results indicated that piperine exhibited anti-proliferative effect in human prostate cancer cells by inducing cell cycle arrest and autophagy.     

Systematic Delivery of Chloroquine and Promethazine Using pH-Sensitive Polymers

Two pH sensitive polymers (Eudragit® L30 D55 and L100) were used as coating materials, respectively, for promethazine hydrochloride and chloroquine phosphate granules formulated with sodium carboxylmethylcellulose and Carbopol 940, respectively, in the ratios 1:1, 1:2, 1:3, and 1:4 (drug:polymer). The granules were characterized. Release studies for the uncoated and coated granules were studied in simulated gastric fluid and simulated intestinal fluid. Result obtained showed that 1:1 and 1:2 ratios of both coated and uncoated granules of the two drugs had short release times and could be recommended for rapid action, whereas 1:4 ratio with low release time could be used for sustained effect. The two granules could be used at varying ratios to obtain desired release characteristics, such that therapeutic concentrations of the two drugs could be achieved.     

Vivax malaria: preliminary observations following a shorter course of treatment with artesunate plus primaquine

The standard adult treatment regimen for Plasmodium vivax malaria is chloroquine (1500 mg over 3 d) plus primaquine (15 or 30 mg daily for 14 d), but patient compliance tends to be poor with the lengthy course. Preliminary observations are reported on the efficacy of a shorter treatment course - artesunate (200mg twice a day for 2 d) plus primaquine (22.5mg base twice a day for 7 d) - given to 28 adult patients infected with P. vivax in Viet Nam. All patients responded quickly to treatment with mean (SD) parasite and fever clearance times of 14.2 (4.0) and 18.6 (8.4) h, respectively. The high dose of primaquine was generally well tolerated, and only one patient (3.6%) had a recurrence of parasitaemia during 28 d of follow-up. As most patients infected with Southeast Asian strains of P. vivax have their first relapse within 28 d after treatment with rapidly eliminated blood schizonticides, the absence of parasitaemia in the remaining 27 patients suggests that this drug regimen was active against both blood and liver stages. Further studies are needed to confirm that this rapidly acting, short artesunate-primaquine regimen can result in better patient compliance and treatment outcomes than the chloroquine-primaquine regimen.     

Pfcrt haplotypes and in-vivo chloroquine response in Sundergarh district, Orissa, India

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) were analyzed as markers of chloroquine (CQ) resistance in the blood samples of patients from two sites of different intensities of malaria transmission (high, n=70; low, n=68) in Sundergarh district of Orissa, India and correlated with the in-vivo response. Early treatment failure (ETF) was significantly more frequent in the high endemic area (32.9 vs. 7.4%, P<0.001), with children below 5 years suffering more. A high frequency of pfcrt K76T mutation was observed in both the areas (87.1 vs. 79.4%, P=0.22). Patients carrying pfcrt 76T were the most likely to develop ETF (odds ratio 36; 95% CI 3.35-1653.3; P<0.001). The ratio of 76T:K76 was 22:9 and 11:14, respectively, in high and low endemic areas (odds ratio 3.1; 95% CI 0.9-11.03; P=0.04), which may be used as a measure of drug pressure. Sequences of pfcrt codons 72-76 showed 16 of the CQ-resistant haplotypes to be SVMNT, 5 CVMNT and 12 CVIET. The CQ-sensitive haplotypes were mostly CVMNK in 10 samples; CVIEK in 2 samples. Both Southeast Asian and South American haplotypes were present, with the latter predominating.     

低浓度氯喹保护成年小鼠视网膜神经节细胞抵抗N-甲基-D-天冬氨酸的兴奋性毒性作用

目的:观察不同浓度氯喹对N-甲基-D-天冬氨酸(NMDA)损伤模型小鼠RGC的保护作用及可能调控机制。方法:健康雄性C57/BL6小鼠54只,随机分为氯喹低浓度组、氯喹高浓度组、PBS对照组,每组均为18只。氯喹低浓度组、氯喹高浓度组小鼠分别按体重10、100 mg/kg剂量腹腔注射氯喹;PBS组小鼠腹腔注射等体积PBS。腹腔注射1次/d,连续2d后,氯喹低浓度组、氯喹高浓度组小鼠左眼玻璃体腔注射5 nmol/L NMDA,对侧眼注射等体积PBS。建模后7d,视网膜铺片RGC染色,计数RGC存活数目;建模后9、10d,分别行视动反应和ERG检查;建模后11 d,行视网膜免疫荧光染色检测各组小鼠视网膜胶质纤维酸性蛋白(GFAP)荧光表达和实时荧光定量PCR(RT-PCR)检测各组小鼠视网膜GFAP、TNF-α、IL-6 mRNA表达。各组间RGC密度、视敏度、光负性反应(PhNR)波振幅比较采用单因素方差分析。结果:建模后7d,与PBS对照组小鼠RGC密度比较,氯喹低浓度组显著升高,差异有统计学意义(F=54.41,P<0.01);氯喹高浓度组降低,但差异无统计学意义(F=1.18,P>0.05)。氯喹低浓度组小鼠视敏度、PhNR波振幅均高于PBS对照组,差异有统计学意义(F=9.10、17.60,P<0.05、<0.01)。氯喹低浓度组小鼠视网膜GFAP绿色荧光表达明显少于PBS对照组、氯喹高浓度组,差异有统计学意义(F=23.66,P<0.05)。低浓度氯喹组小鼠视网膜GFAP(F=110.20)、IL-6(F=167.60)、TNF-α(F=17.78)mRNA表达较高浓度氯喹组、PBS对照组显著下降,差异有统计学意义(P<0.010、<0.001、<0.010)。结论:低浓度氯喹对NMDA损伤所致RGC丢失有保护作用,其作用机制可能与抑制胶质细胞活化与炎症反应有关;高浓度氯喹会加重RGC凋亡。     

海南恶性疟原虫pfcrt等位基因多态性的单体型研究

目的 建立恶性疟原虫pfcrt（Plasmodium falciparum chloroquine resistant transporter）等位基因多态性的单体型的研究方法，比较我国海南省与东南亚及非洲地区恶性疟原虫的pfcrt等位基因多态性的单体型的特征。 方法 来自海南省19份恶性疟滤纸血样提取DNA，通过巢式PCR反应，扩增出包含第72～76、97位密码子的基因片段，根据限制性片段长度多态性（RFLP）分析结果，各选取6份突变型和野生型PCR产物进行DNA测序，检测第72～76、97位密码子序列，从而建立并分析我国海南省恶性疟pfcrt等位基因的单体型。 结果 19份滤纸血样本提取的DNA全部扩增出1条195 bp的片段，酶切消化后，有11份出现1条100 bp左右的酶切片段，为野生型pfcrt等位基因，其余8份为1条195 bp的片段，为突变型。基因序列分析发现，野生型pfcrt等位基因的第72～76位密码子单体型为CVMNK，突变型为CVIET，第97位密码子未发现突变。 结论 我国海南省氯喹抗药性恶性疟原虫pfcrt等位基因第72～76位密码子的单体型与东南亚和非洲地区抗氯喹恶性疟原虫相应单体型一致。     

电离辐射诱导结肠癌细胞自噬以及自噬在辐射中的作用

目的 观察辐射诱导结肠癌SW480细胞发生自噬的情况,以及自噬在辐射中的作用。方法 采用透射电镜观察细胞超微结构,免疫荧光观察自噬泡,蛋白质免疫印迹法检测LC3水平,MTT检测细胞增殖情况,Tanswell小室观察细胞的侵袭,划痕实验观察细胞迁移。结果 重离子与X射线辐射能够诱导SW480细胞发生自噬,且自噬水平随辐射剂量的增加而增加(F=458.526,P<0.05);雷帕霉素(rapamycin, RAPM)能够诱导SW480细胞发生自噬,联合照射时,具有协同作用(F=189.393,P<0.05);氯喹(chloroquine,CQ)能够抑制辐射所诱导的SW480细胞发生的自噬;单纯照射组、照射联合RAPM组细胞的侵袭力、迁移力及活性减弱(F=194.692、629.917、302.903,P<0.05);辐射与CQ联合处理组细胞的侵袭力、迁移力及活性增强(F=194.692、629.917、302.903,P<0.05)。结论 电离辐射可诱导结肠癌SW480细胞发生自噬,自噬对细胞有杀伤作用。     

酮替芬和赛庚啶增强体外培养的恶性疟原虫氯喹抗性株对氯喹反应性的研究

目的 研究酮替芬和赛庚啶增强体外培养的恶性疟原虫氯喹抗性株对氯喹反应性，及其增效机制。 方法 恶性疟原虫氯喹抗性株(Fcc SM1/yN株)取自云南省思茅地区恶性疟患者静脉血，经同步化处理，用新鲜血将红细胞感染率调至0.5%～1.0%，红细胞压积与培养基体积比(红细胞比容)为1 ∶ 9，混匀。制备氯喹药板及氯喹/酮替芬（或氯喹/赛庚啶）组合药板。氯喹药板自上而下每行氯喹终浓度依次为0.312 5～2 560 nmol/L，呈2倍递增。氯喹/酮替芬（或氯喹/赛庚啶）组合药板，是在氯喹药板基础上加入酮替芬（或赛庚啶），每行10孔自左至右终浓度依次为9.77～5 000 nmol/L，呈2倍递增, 每块药板均设A行空白对照。每孔加混匀血样 50 μl，37 ℃ 培养 34 h，镜检计数每200个疟原虫中含3个核以上裂殖体数，计算氯喹单药及各配伍组对恶性疟原虫的半数抑制浓度（IC₅₀），以及酮替芬（或赛庚啶）提高氯喹活性的指数（AEI）。选择AEI值较高的配伍组，进行增效时序性研究。当氯喹对虫体作用 0～10 h 分别加入酮替芬（或赛庚啶），34 h后检测和计算各时间段IC₅₀及AEI。选择氯喹/酮替芬（或氯喹/赛庚啶）最佳配伍剂量，培养恶性疟原虫 20 h，提取总RNA，用实时荧光定量PCR(real-time PCR)分析药物作用前后恶性疟原虫氯喹抗性转移基因（pfcrt）和多药抗性基因（pfmdr1）表达水平。 结果 0.312 5～2 560 nmol/L氯喹与625 nmol/L 酮替芬（或赛庚啶）配伍，增效作用显著，氯喹/酮替芬的IC50为74.53 nmol/L，AEI为0.42；氯喹/赛庚啶的IC50为89.70 nmol/L、AEI为0.30。5 nmol/L氯喹作用 6～7 h 加入625 nmol/L酮替芬（或赛庚啶），增效作用显著，氯喹/酮替芬的IC50为 67.70 nmol/L、AEI为0.47；氯喹/赛庚啶的IC₅₀为81.53 nmol/L、AEI为0.37。5 nmol/L氯喹与625 nmol/L酮替芬（或赛庚啶）配伍，作用20 h，氯喹/酮替芬可使pfcrt基因表达水平升高91%，而氯喹/赛庚啶可使pfmdr1 基因表达水平下降14%。 结论 体外氯喹与适量的酮替芬（或赛庚啶）配伍，能增强恶性疟原虫氯喹抗性株对氯喹的反应性。氯喹对虫体作用 6～7 h 加入酮替芬（或赛庚啶）增效作用显著。该增效作用与pfcrt基因和pfmdr1 基因的表达水平有关。