Da Chengqitang Protects Intestinal Barrier in Septic Mice via Endogenous Antimicrobial Peptide mCRAMP / 中国实验方剂学杂志

ObjectiveMolecular docking and animal experiments were employed to explore the protective effect and mechanism of Da Chengqitang （DCQD） on intestinal barrier in septic mice. MethodText mining method was used to screen the active ingredients in DCQD. AutoDock Tools and Discovery Studio were used to study the interactions of active components with the core target proteins ［claudin-1， tumor necrosis factor （TNF）-α， interleukin （IL）-6， endogenous antimicrobial peptide mCRAMP， Toll-like receptor 4 （TLR4）， and myeloid differentiation primary response gene 88 （MyD88）］ in sepsis. Fifty C57BL/6 mice were randomized into sham， model， low- and high-dose （4 g∙kg-1 and 8 g∙kg-1） DCQD， and ulinastatin groups （n=10）. Before， during， and after the day of modeling surgery， each group was administrated with corresponding drugs. The mice in other groups except the model group were subjected to modeling by cecal ligation and puncture. Enzyme-linked immunosorbent assay （ELISA） was used measure the serum level of D-lactic acid to assess intestinal mucosa permeability. Hematoxylin-eosin staining was employed to observe the histopathological changes in the ileum and assess the intestinal mucosal damage and inflammatory infiltration. Western blotting was employed to determine the expression levels of tight junction proteins claudin-1 and occludin in the ileal tissue， which were indicative of the bowel barrier function. The TNF-α and IL-6 levels were measured by ELISA to assess the intestinal inflammation. The expression of mCRAMP in the ileal tissue was observed by immunohistochemistry. The mRNA levels of mCRAMP， TLR4， and MyD88 in mouse ileal tissue were determined by Real-time polymerase chain reaction， on the basis of which the mechanism of DCQD in protecting the intestinal barrier of septic mice was explored. ResultMolecular docking results showed that most of the 10 active ingredients of DCQD that were screened out by text mining could bind to sepsis targets by van der Waals force， hydrogen bonding， and other conjugated systems. The results of animal experiments showed that compared with the model group， low- or high-dose DCQD lowered the D-lactic acid level in the serum （P<0.01）， alleviated damage to the ileal tissue and mucosal edema， protected the small intestine villus integrity， reduced inflammatory cell infiltration， promoted the expression of claudin-1 （P<0.01）， lowered the IL-6 level （P<0.01）， up-regulated the mRNA and protein levels of mCRAMP （P<0.01）， and down-regulated the mRNA and protein levels of TLR4 and MyD88 （P<0.01） in the ileal tissue. In addition， high-dose DCQD lowered the TNF-α level and promoted the expression of occludin in the ileum tissue （P<0.01）， and low-dose DCQD up-regulated the protein level of occludin in the ileum tissue （P<0.05）. ConclusionDCQD has a protective effect on intestinal barrier in septic mice. It can reduce intestinal inflammation， repair intestinal mucosal damage， improve the tight junction protein level， and reduce intestinal mucosal permeability by up-regulating the mRNA and protein levels of mCRAMP and the down-regulating the expression of genes in the TLR4/MyD88 pathway.

Protective effects and mechanism of Dachengqi decoction on intestinal septic mice / 中国药房

OBJECTIVE To study the protective effects of Dachengqi decoction （DCQD） on intestinal septic mice， and to explore the possible mechanisms from the Toll-like receptor 4（TLR4）/myeloid differentiation factor 88（MyD88） signaling pathway. METHODS The SPF male C57BL/6J mice were randomly divided into Sham group， Sham+DCQD-H group， model （CLP） group， DCQD-L group， DCQD-H group and Positive group. The model of intestinal sepsis was established by cecal ligation and puncture in CLP group， DCQD-L group， DCQD-H group and Positive group. Three days before the operation and seven days after the operation， DCQD-L group and DCQD-H group were given DCQD intragastrically at 4， 8 g/kg （calculated by crude drug）， respectively. Positive group was given ulinastatin intraperitoneally 2 h before operation and 7 d after the operation （at 50 000 U/kg）. In Sham group and Sham+DCQD-H group， only cecum of mice was exposed without ligation and puncture. Sham+DCQD- H group was given DCQD intragastrically （8 g/kg，calculated by crude drug） 3 days before the operation and 7 days after the operation. Both the Sham group and CLP group were given normal saline 0.2 mL intragstrically and intraperitoneally each day， for 10 consecutive days. After the operation， the severity of sepsis was assessed， and the 7 d survival rate of mice was assessed. One hour after the last medication， the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in serum and ileum of mice were determined； the pathological and morphological changes of mice’s liver， lung， kidney and ileum were observed； mRNA expressions of the TLR4 and MyD88 in ileum were tested. RESULTS Compared with CLP group， sepsis score， the levels of TNF-α and IL-6 in serum and ileum （except for IL-6 in ileum of DCQD-L group）， damage score of the liver， lung， kidney and ileum， mRNA expressions of TLR4 and MyD88 in ileum were all decreased significantly in DCQD-L group and DCQD-H group （P＜0.05 or P＜0.01）， while 7 d survival rate （except for DCQD-L group） was increased significantly （P＜0.05）. The damage to liver tissue in mice was significantly improved， and inflammation infiltration and apoptosis were reduced； lung tissue damage had been alleviated， with varying degrees of improvement in alveolar atrophy， bleeding and edema； the renal tissue damage was improved and weakened dilation of renal tubular lumen was weakened； the damage and edema of ileal tissue were significantly improved. CONCLUSIONS DCQD may exert a protective role on intestinal septic model mice. The mechanism may be related to the inhibition of systemic inflammation， the reduction of multiple organ damage， and down-regulation of TLR4/MyD88 signaling pathway.

Study on in vitro microdialysis recovery of hydroxycamptothecine / 中国中药杂志

<p><b>OBJECTIVE</b>To establish the mothod to dectect the microdialysis recovery of HCPT and to investigate the influencing factors, thus to supply experimental basis for in-vivo microdialysis of HCPT.</p><p><b>METHOD</b>The in vitro recovery of HCPT was detected by concentration difference method (increment method and decrement method). The influence of flow rates, medium concentration and temperature on the HCPT recovery and the stability were studied.</p><p><b>RESULT</b>The recovery detected by increment method was the same as by decrement method. The recovery was independent of HCPT concentrations in the medium. The hydroxycamptothecine recovery had good stability and increased as the temperature rose.</p><p><b>CONCLUSION</b>Microdialysis sampling can be used for the pharmacokinetic study of HCPT. Retrodialysis can be used for the determination of the HCPT in vivo recovery.</p>

Pharmacokinetics of nicotine in blood and brain using microdialysis and stable labelled isotope / 中国中药杂志

<p><b>OBJECTIVE</b>Using the stable isotopes as the internal standard of microdialysis technology to establish a new method to study the whole and local brain dynamics of nicotine percutaneous preparations.</p><p><b>METHOD</b>Using th healthy rats as experimental animals, administrating nicotine in abdominal transdermal way, then sample in the blood and brain simultaneously by microdialysis which use deuterium nicotine (DL-nicotine) as internal standard. Detecting the samples by LC-MS/MS method.</p><p><b>RESULT</b>The configuration process in blood and brain both conforms to 2 compartments model, t1/2 is 29.38 min, t1/2beta is 208.51 min, AUC(0-infinity) is 152 127.10 microg x min x L(-1) in the blood t1/2 is 86.64 min, t1/2beta is 386.00 min, AUC(0-infinity) is 152 820.90 microg x min x L(-1) in the brain.</p><p><b>CONCLUSION</b>Dl-nicotine can be used as internal standard of nicotine to correcte the recovery; Stable isotopes internal standard microdialysis technology can be used for studing the whole and the local pharmacokinetic of nicotine and also provide new ideas and methods to studing the process of new drug delivery system.</p>

Study on in vitro microdialysis recovery of Shuanghuanglian / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the feasibility of microdialysis techniqiue to be used in pharmacokinetic study of Chinese medicine, taking Shuanghuanglian as a model drug.</p><p><b>METHOD</b>The samples were obtained by retrodialysis, determined by HPLC gradient elution to calculate the in vitro recovery rate (RR) of specific components. To study the difference of RR of a certain component in different dialysis mediums, and the effect of flow rates and concentration on RR.</p><p><b>RESULT</b>Along with the increase of number of substances in the dialysis medium, the RR of specific components reduced. But the RR was independent of the concentration of the component in the dialysis medium. The RR reduced with the increasing flow rate in the same dialysis medium.</p><p><b>CONCLUSION</b>The microdialysis technique can be used in pharmacokinetics study of Chinese medicine.</p>

Study of pharmacokinetics of nicotine in local brain by using microdialysis and stable labeled isotope / 药学学报

The paper is to report the study of pharmacokinetics of transdermal administered nicotine in the brain of freely moving rat by using microdialysis with stable labeled isotope as internal standard. The pharmacokinetic behavior of nicotine in Sprague Dawley rat brain was investigated after intranasal administration (3.75 mg). Brain fluid samples were collected by intracerebral microdialysis with DL-nicotine as internal standard. Concentrations of nicotine and DL-nicotine in the sample were measured by HPLC-MS/MS. Main pharmacokinetic parameters were calculated and analyzed by Das 2.0 pharmacokinetic software. The recovery of nicotine and the delivery of DL-nicotine were the same. The fate of absorption and distribution was two compartment model and the values of t1/2alpha was 170.31 min, t1/2beta was 263.30 min and the AUC(0-infinity) was 2.75 x 10(5) microg x L(-1) min separately. DL-nicotine can be used to calibrate the recovery of nicotine, and the new method of stable isotope microdialysis can be used to study the pharmacokinetics of freely moving rat. It will make sense for the treatment of addiction of tobacco and provide a new thought for the research of pharmacokinetics-pharmacodynamic combination.

Internal standard method to determine the recovery of nicotine in microdialysis / 药学学报

The paper reports the evaluation of the feasibility of using internal standard method for the determination of nicotine recovery in microdialysis in vitro. This in vitro experiment included two conditions. Nicotine and codeine phosphate were dissolved in Ringer's solution. Nicotine, codeine phosphate and the mixture of them were perfused through the CMA30 linear probe separately to calculate the proportion of the recovery (or delivery) of nicotine to that of codeine phosphate. And then codeine was perfused through the probe which was immersed in nicotine solution with different concentrations to calculate the proportion, too. In another condition nicotine was dissolved in rat plasma. The rat plasma protein binding rate was determined by using retrodialysis and internal standard method in vitro. The results are as follows: the proportion of the recovery (or delivery) of nicotine to that of codeine phosphate was fairly stable. The delivery of codeine was independent of nicotine concentration in the external medium. Protein binding rate determined by retrodialysis was almost the same as that determined by internal standard method. It suggests that the internal standard method is an effective way in the determination of nicotine recovery and codeine phosphate can be used as the internal standard.

Study on optimization of formulation and preparation process of sinomenine liposomes / 中国中药杂志

<p><b>OBJECTIVE</b>To optimize the formulation and preparation process of sinomenine liposomes.</p><p><b>METHOD</b>Method of aether injection and mixture uniform design were adopted to determine the formulation of sinomenine liposomes is the proportion of phospholipids, cholesterol and Vitamin E with the index of entrapment efficiency. And the single-factor test was used to study the preparation process of the liposomes, including the volume of buffer solution, the preparation temperature and the ultrasonic time.</p><p><b>RESULT</b>The optimized formulation was that the ratio of sinomenine : phospholipids : cholesterol : vitamin E mass ratio was 8.92 : 60.35 : 28.81 : 1.91. The volume of buffer solution was 50 mL x g(-1) membrane, the preparation temperature was 50 degrees C, and the ultrasonic time was 20 min.</p><p><b>CONCLUSION</b>Satisfactory shape and entrapment efficiency of the liposomes can be obtained by the optimized formulation and preparation process.</p>

Pharmacokinetic Studies of Sinomenine by Blood Microdialysis Technique / 广州中医药大学学报

[Objective] To explore the feasibility and advantages of blood microdialysis technique for the pharmacokinetic study of sinomenine. [Methods] In this study, rats served as the experimental subjects and sinomenine was administered to the rats by intravenous injection. With microdialysis technique for blood sampling, the blood concentration of sinomenine was determined by high performance liquid chromatography (HPLC) and the associated pharmacokinetic parameters were analyzed by 3p97 program. [Results] The pharmacokinetics process of sinomenine in rats presented as the two-compartment model. The half-life of distribution phase was 10.98min and that of elimination phase was 44.71 min. [Conclusion] It is feasible to study pharmacokinetic parameters of sinomenine by using blood microdialysis technique. In comparison with the traditional technique, blood microdialysis technique is superior to the examination of drug blood concentration .

In - Vitro Experiment for Detecting Microdialysis Recovery rate of Sinomenine / 广州中医药大学学报

[Objective] To explore the in-vitro determination method for the recovery rate of sinomenine in microdialysis probe and to investigate the influencing factors. [Methods] The recovery rate of sinomenine was detected by concentration difference method (by gain and by loss) and zero-net flux method. [Results] The recovery rate detected by increment method was as the same as that by decrement method; the recovery rate had no correlation with the sinomenine concentration in the solvent. Sinomenine recovery assessed by concentration difference method had a good intra-day stability and intra-day reproducibility. Sinomenine concentration and recovery in the solvent could be determined accurately by zero-net flux method. [ Conclusion ] Microdialysis sampling can be used for the pharmacokinetic study of sinomenine and decrement method (ie. retrodialysis) can be used for the determination of recovery rate of sinomenine.

Study on the In-vitro Microdialysis Recovery of Nicotine and Its Influence Factors / 广州中医药大学学报

Objective To detect the in-vitro microdialysis recovery of nicotine by decrement method(i.e.retrodialysis) and to investigate its influence factors,thus to supply experimental evidence for the in-vivo microdialysis of nicotine.Methods The in-vitro recovery of nicotine was detected by concentration difference method(increment method and decrement method).The stability of recovery was observed.The effects of the pretreatment with perfusate,and the flow rates and concentrations of the perfusate on nicotine recovery were studied.Results Filtering and stirring treatment increased the recovery,but the time of ultrasound degassing showed no effect on the recovery.The recovery detected by increment method was as the same as that detected by decrement method.Nicotine recovery was independent of nicotine concentration in the external medium,showing good stability.Conclusion It is necessary to filter and degas the perfusate before microdialysis.Retrodialysis can be used for the determination of in-vivo nicotine recovery.