Intestinal injury can be reduced by intra-arterial postischemic perfusion with hypertonic saline

AIM:To investigate the effect of local intestinal perfusion with hypertonic saline(HTS) on intestinal ischemia-reperfusion injury(IRI) in bothex vivo andin vivo rat models.METHODS:All experiments were performed on male Wistar rats anesthetized with pentobarbital sodium given intraperitoneally at a dose of 60 mg/kg.Ex vivo vascularly perfused rat intestine was subjected to 60-min ischemia and either 30-min reperfusion with isotonic buffer(controls),or 5 min with HTS of 365 or 415 mOsm/L osmolarity(HTS 365mOsm or HTS 415mOsm,respectively) followed by 25-min reperfusion with isotonic buffer.The vascular intestinal perfusate flow(IPF) rate was determined by collection of the effluent from the portal vein in a calibrated tube.Spontaneous intestinal contraction rate was monitored throughout.Irreversible intestinal injury or area of necrosis(AN) was evaluated histochemically using 2.3.5-triphenyltetrazolium chloride staining.In vivo,30-min ischemia was followed by either 30-min blood perfusion or 5-min reperfusion with HTS 365mOsm through the superior mesenteric artery(SMA) followed by 25-min blood perfusion.Arterial blood pressure(BP) was measured in the common carotid artery using a miniature pressure transducer.Histological injury was evaluated in both preparations using the Chui score.RESULTS:Ex vivo,intestinal IRI resulted in a reduction in the IPF rate during reperfusion(P < 0.05 vs sham).The postischemic recovery of the IPF rate did not differ between the controls and the HTS 365mOsm group.In the HTS 415mOsm group,postischemic IPF rates were lower than in the controls and the HTS 365mOsm group(P < 0.05).The intestinal contraction rate was similar at baseline in all groups.An increase in this parameter was observed during the first 10 min of reperfusion in the control group as compared to the sham-treated group,but no such increase was seen in the HTS 365mOsm group.In controls,AN averaged 14.8% ± 5.07% of the total tissue volume.Administration of HTS 365mOsm for 5 min after 60-min ischemia resulted in decrease in AN(5.1% ± 1.20% vs controls,P < 0.01).However,perfusion of the intestine with the HTS of greater osmolarity(HTS 415mOsm) failed to protect the intestine from irreversible injury.The Chiu score was lower in the HTS 365mOsm group in comparison with controls(2.4 ± 0.54 vs 3.2 ± 0.44,P = 0.042),while intestinal perfusion with HTS 415mOsm failed to improve the Chiu score.Intestinal reperfusion with HTS 365mOsm in the in vivo series secured rapid recovery of BP after its transient fall,whereas in the controls no recovery was seen.The Chiu score was lower in the HTS 365mOsm group vs controls(3.1 ± 0.26 and 3.8 ± 0.22,P = 0.0079 respectively,),although the magnitude of the effect was lower than in the ex vivo series.CONCLUSION:Brief intestinal postischemic perfusion with HTS 365mOsm through the SMA followed by blood flow restoration is a protective procedure that could be used for the prevention of intestinal IRI.

Intestinal permeability of metformin using single-pass intestinal perfusion in rats

AIM： To characterize the intestinal transport and mechanism of metformin in rats and to investigate whether or not metformin is a substrate for P-glycoprotein （P-gp）. METHODS： The effective intestinal permeability of metformin was investigated using single-pass intestinal perfusion （SPIP） technique in male Waster rats. SPIP was performed in three isolated intestinal segments （duodenum, jejunum and ileum） at the same concentration of metformin （50 μg/mL） to test if the intestinal transport of metformin exhibited site- dependent changes, and in a same isolated intestinal segment （duodenal segment） at three different concentrations of metformin （10, 50, 200 μg/mL） to test if the intestinal transport of metformin exhibited concentration-dependent changes. Besides, P-gp inhibitor verapamil （400 μg/mL） was co-perfused with metformin （50 μg/mL） in the duodenum segment to find out if the intestinal absorption of metformin was affected by P-gp exiting along the gastrointestinal track. Stability studies were conducted to ensure that the loss of metformin could be attributed to intestinal absorption. RESULTS： The effective permeability values （Peff） of metformin in the jejunum and ileum at 50μg/mL were significantly lower than those in the duodenum at the same concentration. Besides, Peff values in the duodenum at high concentration （200 μg/mL） were found to be significantly lower than those at low and medium concentrations （10 and 50 μg/mL）. Moreover the coperfusion with verapamil did not increase the Peff value of metformin at 50 μg/mL in the duodenum.CONCLUSION： Metformin could be absorbed from the whole intestine, with the main absorption site at duodenum. This concentration-dependent permeability behavior in the duodenum indicates that metformin is transported by both passive and active carrier-mediated saturable mechanism. The Peff value can not be increased by co-perfusion with verapamil, indicating that absorption of metformin is not efficiently transported by P-gp in the gut wall. Furthermore metformin is neither a substrate nor an inducer of P-gp. Based on the Peff values obtained in the present study and using established relationships, the human fraction dose absorbed for metformin is estimated to be 74%-90% along human intestine.

The preparation of Garcinia Glycosides solid dispersion and intestinal absorption by rat in situ single pass intestinal perfusion

Garcinia Glycosides is a candidate drug obtained by structural modification of Gambogic Acid (GA), which was acquired through High Throughput Screening(HTS). As Garcinia Glycosides is an effective but insoluble anti-tumor drug, the aim of this study was to obtain a solid dispersion form Garcinia Glycosides by using solvent-melt method so that improve the solubility and dissolution rate. The solid dispersion was characterized by High Performance Liquid Chromatography (HPLC), infrared spectroscopy and evaluated the intestinal absorption of the drug by rat in situ single pass intestinal perfusion. The results showed the increase of solubility, dissolution velocity and absorption compared to other forms. This indicated that solid dispersion could greatly improve the relative bioavailability of Garcinia Glycosides in vivo.

Motility patterns of ex vivo intestine segments depend on perfusion mode

AIM:To evaluate and characterize motility patterns from small intestinal gut segments depending on different perfusion media and pressures.METHODS:Experiments were carried out in a custom designed perfusion chamber system to validate and standardise the perfusion technique used.The perfusion chamber was built with a transparent front wall allowing for optical motility recordings and a custom made fastener to hold the intestinal segments.Experiments with different perfusion and storage media combined with different luminal pressures were carried out to evaluate the effects on rat small intestine motility.Software tools which enable thevisualization and characterization of intestinal motility in response to different stimuli were used to evaluate the videotaped experiments.The data collected was presented in so called heatmaps thus providing a concise overview of form and strength of contractility patterns.Furthermore,the effect of different storage media on tissue quality was evaluated.HaematoxylinEosin stainings were used to compare tissue quality depending on storage and perfusion mode.RESULTS:Intestinal motility is characterized by different repetitive motility patterns,depending on the actual situation of the gut.Different motility patterns could be recorded and characterized depending on the perfusion pressure and media used.We were able to describe at least three different repetitive patterns of intestinal motility in vitro.Patterns with an oral,anal and oro-anal propagation direction could be recorded.Each type of pattern finalized its movement with or without a subsequent distension of the wavefront.Motility patterns could clearly be distinguished in heatmap diagrams.Furthermore undirected motility could be observed.The quantity of the different patterns varies and is highly dependent on the perfusion medium used.Tissue preservation varies depending on the perfusion medium utilized,therefore media with a simple composition as Tyrode solution can only be recommended for short time experiments.The more complex media,MEM-HEPES medium and especially AQIXRS-I tissue preservation reagent preserved the tissue much better during perfusion.CONCLUSION:Perfusion media have to be carefully chosen considering type and duration of the experiments.If excellent tissue quality is required,complex media are favorable.Perfusion pressure is also of great importance due to the fact that a minimum amount of luminal pressure seems to be necessary to trigger intestinal contractions.

Absorption of Curcumin Monophosphate in Rat Intestinal Circulation

[Objectives] This study aimed to study the absorption of curcumin monophosphate, a derivative of curcumin, in the small intestine of rats. [Methods] In situ recirculation perfusion technique was used to study the absorption of curcumin monophosphate in intestinal circulation of rats, and the content of curcumin monophosphate in intestinal circulation solution was determined by high-performance liquid chromatography/ultraviolet detector(HPLC/UV). [Results] The established HPLC/UV method has good specificity. Linear regression was conducted between the peak area of curcumin monophosphate(A) and the concentration of curcumin monophosphate(C). The established standard curve equation was y=30.07x+102.48(R^2=0.999 0), indicating that curcumin monophosphate has good linearity in the range of 1.25-100.00 μg/mL. After excluding the degradation of the drug and the loss of sampling, within the first 2 h of the experiment, the drug absorption in the small intestine of rat was 1.39 mg, accounting for 97.2% of the total drug absorption during the 5-h experimental period, and the absorption rate was 53.9%.[Conclusions] Curcumin monophosphate has a good absorption in the small intestine of rats.

Absorption characteristics of the total alkaloids from Mahonia bealei in an in situ single-pass intestinal perfusion assay

AIM: To investigate the absorption characteristics of the total alkaloids from Mahoniae Caulis(TAMC) through the administration of monterpene absorption enhancers or protein inhibitors. METHOD: The absorption behavior was investigated in an in situ single-pass intestinal perfusion(SPIP) assay in rats. RESULTS: The intestinal absorption of TAMC was much more than that of a single compound or a mixture of compounds(jatrorrhizine, palmatine, and berberine). Promotion of absorption by the bicyclic monoterpenoids(borneol or camphor) was higher than by the monocyclic monoterpenes(menthol or menthone), and promotion by compounds with a hydroxyl group(borneol or menthol) was higher than those with a carbonyl group(camphor or menthone). The apparent permeability coefficient(Papp) of TAMC was increased to 1.8-fold by verapamil, while it was reduced to one half by thiamine. The absorption rate constant(Ka) and Papp of TAMC were unchanged by probenecid and pantoprazole. CONCLUSION: The intestinal absorption characteristics of TAMC might be passive transport, and the intestinum tenue was the best absorptive site. In addition, TAMC might be likely a substrate of P-glycoprotein(P-gp) and organic cation transporters(OCT), rather than multidrug resistance protein(MRP) and breast cancer resistance protein(BCRP). Compared with a single compound and a mixture of compounds, TAMC was able to be absorbed in the blood circulation effectively.

肠脂肪酸结合蛋白与心源性休克患者的预后相关性分析

目的探讨血清肠脂肪酸结合蛋白(IFABP)对重症医学科(ICU)心源性休克患者预后的预测。方法选择2020年1月至2022年5月ICU心源性休克患者99例。入ICU24h、3d采用酶联免疫吸附试验(ELISA)测定血清IFABP浓度,根据28d生存情况分为存活组和死亡组,比较两组患者IFABP浓度变化及对生存预后的影响。结果心源性休克患者28 d存活62例,存活率62.6%,存活组患者APAECH II评分及SOFA评分低于死亡组、呼吸机使用和血管活性药物比例低于死亡组,差异有统计学意义(P<0.05)。存活组入ICU 24 h、3 d血清IFABP浓度均低于死亡组(P<0.05)。Logistic回归分析显示,入ICU24 h血清IFABP水平、APACHE II评分及SOFA评分为影响心源性休克患者28 d死亡的危险因素(P<0.05)。ROC曲线分析IFABP对心源性休克患者预后有更好的预测价值,敏感性83.8%、特异性61.3%。结论IFABP血清水平对心源性休克患者28d生存有预测价值,血清IFABP浓度升高与心源性休克患者存活率下降有关。

异功散中人参皂苷类成分在脾虚大鼠模型肠吸收动力学变化及其陈皮的影响

目的基于脾虚大鼠模型探讨异功散中陈皮对人参皂苷类成分肠吸收变化的影响及其影响是否与P-gp蛋白外排作用有关。方法分组设计加入P-gp蛋白激动剂利福平与抑制剂维拉帕米,将36只SD大鼠随机分为空白对照组(6只)与模型组(30只),予以皮下注射利血平建立脾虚模型后将模型组分为异功散组(Y组)、去陈皮异功散组(Y-C组)、异功散利福平组(Y+R组)、去陈皮异功散利福平组(Y-C+R组)、去陈皮异功散维拉帕米组(Y-C+V组),以人参皂苷Rb1、人参皂苷Re及人参皂苷Rg1为指标成分,进行在体单向肠灌流实验,采用HPLC-MS测定指标成分含量并计算肠吸收动力学参数有效渗透系数(P_(eff))与吸收速率常数(K_(a))。结果与Y组相比,Y-C组中人参皂苷Rb1和人参皂苷Re在多个灌流时段的P_(eff)显著降低,Y+R组中人参皂苷Rb1、人参皂苷Re及人参皂苷Rg1的P_(eff)和K_(a)在多个灌流时段降低,表明陈皮促进人参皂苷Rb1和人参皂苷Re肠吸收且该作用与P-gp蛋白活性有关,但未能影响人参皂苷Rg1的肠吸收;与Y-C组相比,Y-C+V组中P-gp蛋白抑制剂维拉帕米使人参皂苷Rb1和人参皂苷Re在多个灌流时段P_(eff)显著升高,而Y-C+R组中P-gp蛋白激动剂利福平使人参皂苷Rb1、人参皂苷Re及人参皂苷Rg1的P_(eff)和K_(a)在多个灌流时段显著降低,说明无陈皮干预下,人参皂苷Rb1、人参皂苷Re及人参皂苷Rg1也可能是P-gp蛋白的底物。结论异功散中陈皮通过发挥抑制P-gp蛋白活性的类维拉帕米作用来促进人参皂苷Rb1和人参皂苷Re肠吸收。

在体单向肠灌流模型测定甘草素的药物渗透性及生物药剂学分类预测

目的测定不同浓度甘草素在大鼠不同肠段的渗透性,初步探讨甘草素在肠道中的吸收机制,并对甘草素进行生物药剂学分类预测。方法参考2020年版《中国药典》中关于溶解度定义,考察甘草素在pH 1.2、4.0、6.8、7.4的缓冲液和水中的平衡溶解度来评价甘草素的溶解性,采用在体单向肠灌流模型测定甘草素在25、50、75μg·mL^(-1)对大鼠十二指肠、结肠、空肠、回肠的渗透性;结合溶解性和渗透性结果预测甘草素生物药剂学分类。结果甘草素在不同pH下和水中平衡溶解度都低于100μg·mL^(-1),溶解度为不溶或几乎不溶,亲脂性不强,甘草素在大鼠不同肠段下的渗透性都大于1.2×10^(-3)cm·min^(-1),为易吸收药物。结论甘草素在大鼠肠吸收中存在自身吸收抑制,存在吸收饱和特性,可能有主动转运或扩散等转运机制参与,预测甘草素为生物药剂学Ⅱ类药物,为低溶解度,高渗透性药物。

在体单向肠灌流法研究知母提取物肠吸收特性

目的:研究知母提取物7个活性成分在大鼠肠道的吸收特性,揭示知母提取物活性成分的肠吸收机制。方法:选用大鼠单向肠灌流模型进行知母7种活性成分的肠吸收实验,采用固相萃取法处理肠灌流样品,通过LCMS/MS定量方法测定知母多种活性成分的吸收变化。结果:从Ka来看,芒果苷、新芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ以及菝葜皂苷元的Ka存在显著性差异(P<0.05),提示这些物质主要吸收部位在空肠。知母提取物中主要有效成分P_(eff)值均>1.0×10^(-3)cm/min,提示各成分在大鼠十二指肠段和空肠的吸收均较好。结论:芒果苷、新芒果苷、知母皂苷BⅡ、宝藿苷Ⅰ以及菝葜皂苷元的主要吸收部位在空肠。

丹酚酸B大鼠在体肠吸收研究

目的:研究丹酚酸B在大鼠小肠的吸收情况。方法:采用在体单向灌流法进行小肠吸收实验,利用HPLC法测定灌流液中丹酚酸B的浓度。结果:丹酚酸B浓度为24.63μg·mL-1时,在十二指肠、空肠、回肠段的吸收速率常数(Ka)和表观吸收系数(Papp)无显著性差异(P>0.05)。与12.54μg·mL-1浓度比较,48.12μg·mL-1浓度时Sal B在空肠的Ka和Papp均显著降低[Ka:(2.13±0.50)×10-2vs(3.10±0.42)×10-2min-1,P<0.05;Papp:(2.07±0.49)×10-3vs(3.10±0.51)×10-3cm.min-1,P<0.05]。结论:丹酚酸B的小肠吸收存在高浓度饱和现象,且在全肠道吸收良好,且在小肠内无特定吸收部位,提示该药物适合制成日服2次的缓控释制剂。

大鼠在体肠灌流模型中补骨脂素的吸收研究

目的:研究补骨脂素在大鼠小肠的吸收特性及其机制,探讨其在临床上血药浓度差异较大的原因。方法:采用改良大鼠在体单向肠灌流模型,通过颈静脉插管补充血液、肠系膜静脉插管采集血样,同时收集灌流流出液,以反相高效液相色谱法测定补骨脂素含量,计算其通透率。结果:补骨脂素在大鼠小肠内通透率较高,吸收较好。不同浓度的补骨脂素灌流时均以约30%的比例吸收入血,且通透率Plumen,Pblood在所测浓度范围内基本保持不变。结论:补骨脂素在大鼠小肠的转运机制为被动转运,不受P-糖蛋白的影响,临床上血药浓度个体差异大可能与肝脏的首过效应有关,而非小肠吸收代谢所致。

隐丹参酮在小肠吸收机制的实验研究

目的 研究隐丹参酮在大鼠小肠的吸收机制。方法 用大鼠在体一过式肠灌流方法,通过肠系膜静脉插管采集血样,同时收集灌流流出液,以高效液相色谱法测定样品中隐丹参酮含量,计算其通透率。结果 隐丹参酮在大鼠小肠的肠管通透率(Plumen)和血管通透率(Pblood)随浓度的增加而下降;加入P-糖蛋白抑制剂维拉帕米后其通透率增高。结论 隐丹参酮在大鼠小肠的转运机制可能为主动转运,隐丹参酮可能为P-糖蛋白底物。

长春西汀的大鼠在体肠吸收研究

采用单向灌流技术考察长春西汀的大鼠在体肠吸收性质。结果表明,灌流速度对药物吸收速率常数(Ka)和表观吸收系数(Papp)有极显著影响(P<0.01);药物浓度对Ka和Papp无显著影响(P>0.05);药物在全肠道吸收较好,吸收窗主要在小肠,且小肠内无明显的特定吸收部位;羟丙基-β-环糊精在1%～4%范围内对药物的肠吸收无显著影响。

磷脂对甘草酸二铵小肠吸收的影响

研究磷脂对甘草酸二铵及甘草次酸小肠吸收的影响。建立大鼠原位单向肠灌流及伴有肠系膜插管的肠灌流模型,采用高效液相色谱法检测灌流液及血浆中的药物浓度,计算表观通透系数Papp和吸收速率常数Ka。建立大鼠灌胃后肝门静脉取血模型,测定门静脉血浆中药物浓度,以对灌流结果进一步验证。甘草酸二铵和甘草次酸均能跨肠壁吸收,其Papp分别为0.36和5.73 cm.min-1,加入磷脂后,Papp分别提高到0.68和7.98 cm.min-1。磷脂使甘草酸二铵本身的吸收略有增加,但统计学检验差异不显著,但对其肠道菌群代谢物甘草次酸的吸收有显著促进作用。

灯盏花素磷脂复合物改善大鼠小肠吸收的研究

目的研究灯盏花素磷脂复合物对灯盏花素实验性小肠吸收的改善作用。方法采用大鼠在体小肠循环法和离体外翻肠囊法研究灯盏花素普通粉剂和灯盏花素磷脂复合物的小肠吸收及肠壁通透性。结果大鼠在体小肠吸收实验中,灯盏花素磷脂复合物单位吸收量和单位吸收率分别提高7375%和4711%,肠壁通透性增加7313%;离体小肠吸收实验中,灯盏花素磷脂复合物单位吸收量和单位吸收率分别提高4121%和30%,肠壁通透性增加3935%;在0～100μg·mL-1内,吸收符合Fick’s扩散定律,表现出一级动力学过程。结论灯盏花素磷脂复合物主要以被动扩散的方式吸收,并通过提高肠壁通透性来改善其吸收。

5种大黄游离蒽醌大鼠在体肠吸收的单向灌流法研究

目的研究5种大黄游离蒽醌混合物(FAM,芦荟大黄素、大黄酚、大黄素、大黄酸、大黄素甲醚)在大鼠各肠段的吸收状况。方法运用大鼠在体单向灌流肠吸收模型,HPLC法测定灌流液中FAM的浓度,分别考察FAM中5种物质在十二指肠、空肠、回肠、结肠及胆汁引流十二指肠段的吸收速率常数(Ka)和药物表观吸收系数(Papp)。结果芦荟大黄素、大黄素和大黄酸主要吸收部位为十二指肠,大黄酚在十二指肠和结肠段的吸收均较大,大黄素甲醚主要吸收部位为结肠;各物质在回肠段的吸收速率均最低(P<0.05)。结论 FAM作为肠灌流液时,各物质的吸收与肠道的酸碱内环境和物质本身的脂溶性有关,同时可能存在相互间吸收的竞争与拮抗作用。胆汁对FAM在十二指肠的吸收有抑制作用。单向肠灌流实验表明FAM在各肠段吸收良好,这与体循环血液中除大黄酸外各物质测定浓度较低存在矛盾,推测这与相关物质的肠道代谢有关。

对大鼠在体肠单向灌流技术中重量法的评价

目的:评价重量法在大鼠在体肠单向灌流技术中的应用。方法:以长春西汀为模型药物,采用大鼠在体肠单向灌流技术,对重量法和酚红法计算的净水流量值(NWF)、药物吸收速率常数(Ka)和药物表观吸收系数(Papp)进行比较,并对重量法中收集液在密度修正前后的NWF和Papp值进行比较。结果:重量法计算所得的NWF值显著高于酚红法(P<0.05),前者计算所得的Ka和Papp值略高于后者(P>0.05);重量法能显著减少实验误差;对收集液密度进行修正可以进一步提高实验数据的准确性。结论:重量法可以作为大鼠单向灌流在体肠吸收实验中校正灌流液体积的有效方法,它避免了加入“标示物”给实验带来的一系列问题。

蟾毒灵的大鼠在体肠吸收机制研究

目的研究蟾毒灵的大鼠在体肠吸收机制。方法采用大鼠在体肠单向灌流吸收实验模型，并应用重量法校正灌流液体积，考察吸收部位、药物浓度、pH值及P-糖蛋白（P-gp）抑制剂对药物吸收的影响。结果蟾毒灵的吸收速率常数（Ka）和表观吸收系数（Papp）分别按照空肠、回肠、十二指肠、结肠的顺序下降。220μg·ml-1的药物浓度对K和Papp无显著影响（P〉0．05）；灌流液pH值对药物吸收有影响；含P—gP抑制剂组与不合P—gP抑制剂组相比，Ka和Papp无显著性差异（P〉0．05）。结论蟾毒灵为全肠段吸收，吸收机制为被动扩散，且P—gP对药物的小肠吸收基本无影响。

单向灌流法研究磷酸川芎嗪的大鼠在体肠吸收

目的研究磷酸川芎嗪的大鼠在体肠吸收性质。方法运用单向灌流模型、采用HPLC法对药物的质量浓度进行检测,分别研究灌流速度、药物质量浓度、pH值以及吸收部位对磷酸川芎嗪吸收的影响。结果灌流速度和pH值对磷酸川芎嗪的吸收速率常数(ka)和表观吸收系数(Papp)有显著性影响;药物质量浓度对ka和Papp无显著性影响;小肠各段间药物吸收的ka和Papp无显著性差异,但与回肠段相比吸收明显增大。结论磷酸川芎嗪的吸收速率不受药物质量浓度的影响,而与灌流速度、灌流液的pH值和肠段部位有关。药物在全肠道吸收较好,吸收窗主要在小肠,且小肠内无明显的特定吸收部位。