Investigation of the Sensitive Target Cell of Spermatocytes Injury Induced by Triptolide in Mice Testes / 中国药学杂志

OBJECTIVE: To investigate the sensitive target cell population of spermatocyte injury induced by TL in mice. METHODS: Seven to eight-week-old healthy male C57BL/6 mice were orally administered triptolid(TL) of 0.125, 0.25 and 0.5 mg•kg-1 per day, and dissected on days 3, 7, 11 and 15 respectively. The extracted testes were fixed with formaldehyde. Paraffin sections and hematoxylin-eosin (HE) staining were performed to determine the optimal time point and dose level to be applied for sensitive target cell population analysis of spermatocyte injury induced by TL. The primary spermatocytes in different stages were clearly distinguished and counted based on the characteristic distribution profile of γ-H2AX in spermatocytes under immunohistochemical staining. The sensitive target cell populations of spermatocyte injury were determined according to the decreased percentage of spermatocytes in different stages. RESULTS: HE staining showed that the best dose-effect relationship of spermatogenic cell injury in the testes was present on day 11 after TL administration (The severity of the lesion ranged from a minimal degree in the 0.125 mg•kg-1 group to a mild to moderate injury in the 0.25 mg•kg-1 group, and finally to a marked injury in the 0.5 mg•kg-1 group). The degree of injury in the 0.125 and 0.25 mg•kg-1 groups was appropriate and suitable for determination of sensitive target cell populations. γ-H2AX immunohistochemical staining indicated that the γ-H2AX showed different distribution characteristics in nucleus in different stages of spermatocyte differentiation: scattered throughout the nucleus in a few discrete foci to fill the whole nuclear in leptotene; assembled in the chromatin regions in zygotene; located on the edge of the nucleus in a single foci in the pachytene; located in the nucleus in a single foci in the diplotene. The counting results showed that the absolute number of primary spermatocytes in all differentiating stages decreased slightly without statistical significance (P>0.05) in the 0.125 mg•kg-1 dose group; the absolute number of primary spermatocytes decreased significantly with statistical significance (P<0.01 or 0.001) in the 0.25 mg•kg-1 dose group. There was higher decreased percentage of the leptotene primary spermatocyte among the differentiating stages before pachytene stage and with statistical significance (P<0.05) at 0.25 mg•kg-1 when compared with the pachytene primary spermatocyte. CONCLUSION: γ-H2AX immunohistochemical staining can clearly distinguish primary spermatocytes at different stages. The leptotene primary spermatocytes are the most likely sensitive target cells in the testicular spermatocyte-injury induced by TL administration.

Facile one-pot synthesis of a aptamer-based organic-silica hybrid monolithic capillary column by "thiol-ene" click chemistry for detection of enantiomers of chemotherapeutic anthracyclines.

In the current study, we developed a facile strategy for the one-pot synthesis of an aptamer-based organic-silica hybrid monolithic capillary column. A 5'-SH-modified aptamer, specifically targeting doxorubicin, was covalently modified in the hybrid silica monolithic column by a sol-gel method combined with "thiol-ene" click reaction. The prepared monolithic column had good stability and permeability, large specific surface, and showed excellent selectivity towards chemotherapeutic anthracyclines of doxorubicin and epirubicin. In addition, the enantiomers of doxorubicin and epirubicin can be easily separated by aptamer-based affinity monolithic capillary liquid chromatography. Furthermore, doxorubicin and epirubicin spiked in serum and urine were also successfully determined, which suggested that the complex biological matrix had a negligible effect on the detection of doxorubicin and epirubicin. Finally, we quantified the concentration of epirubicin in the serum of breast cancer patients treated with epirubicin by intravenous injection. The developed analytical method is cost-effective and rapid, and biological samples can be directly analyzed without any tedious sample pretreatment, which is extremely useful for monitoring medicines in serum and urine for pharmacokinetic studies.

Quantitative study of cellular heterogeneity in doxorubicin uptake and its pharmacological effect on cancer cells.

Cellular heterogeneity in doxorubicin (DOX) uptake and its relationship with pharmacological effect on cancer cells were quantitatively investigated for the first time. An in vitro experimental model was established by treating human leukemia K562 and breast cancer MCF-7 cells with different schedules of DOX with or without surface P-glycoprotein (P-gp) inhibitor verapamil (VER). The cellular heterogeneity in DOX uptake was quantitatively examined by single-cell analysis using capillary electrophoresis coupled with laser-induced fluorescence detection. The corresponding cytotoxic effect was tested by cellular morphology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium and flow cytometry assays. The expression of cellular membrane surface P-gp was determined by flow cytometry. Results showed that the cellular heterogeneity exists in DOX uptake. The single-high DOX schedule leads to lower uptake heterogeneity and higher mean drug uptake. The cellular heterogeneity in DOX uptake was found to be negatively correlated with drug cytotoxicity and surface P-gp expression, with r = -0.7680 to ~ -0.9587. VER reduces the cellular variation in DOX uptake, suggesting that surface P-gp may be one of the causes of the cellular heterogeneity in DOX uptake. This research demonstrates the importance of quantitative study of cellular heterogeneity in drug uptake and its potential application in drug schedule design, response prediction and therapy modulation.

Preparation approaches of the coated capillaries with liposomes in capillary electrophoresis.

The use of liposomes as coating materials in capillary electrophoresis has recently emerged as an important and popular research area. There are three preparation methods that are commonly used for coating capillaries with liposomes, namely physical adsorption, avidin-biotin binding and covalent coupling. Herein, the three different coating methods were compared, and the liposome-coated capillaries prepared by these methods were evaluated by studying systematically their EOF characterization and performance (repeatability, reproducibility and lifetime). The amount of immobilized phospholipids and the interactions between liposome or phospholipid membrane and neutral compounds for the liposome-coated capillaries prepared by these methods were also investigated in detail. Finally, the merits and disadvantages for each coating method were reviewed.

A novel covalent coupling method for coating of capillaries with liposomes in capillary electrophoresis.

A novel covalent coupling method for coating of capillaries with liposomes has been developed, which includes three steps: (i) epoxy-diol coating, (ii) activation with 2,2,2-trifluoroethanesulfonyl chloride, and (iii) liposome coupling. The coating conditions, such as the reaction time and temperature of liposome coupling, the content of dimyristoylphosphatidylethanolamine in liposomes, were optimized. Vesicles were visualized on the inner silica wall as confirmed by atomic force microscopy. The effectiveness of the coating was demonstrated by investigating the effect of pH of BGE on EOF and separating neutral compounds. The intra- and inter-capillary variations in EOF are 4.02% RSD (n=30) and 6.72% RSD (n=4) respectively, and the coated capillaries can be used to perform analysis at least for one month without any performance deterioration when stored at 4 degrees C. A set of drugs with diverse structures was applied into the developed liposome-coated CE. The normalized capacity factor (K) was introduced to quantitatively evaluate drug-membrane interactions. The relationship between log K and the fraction dose absorbed in humans (Fa%) shows that the liposome-coated CE can be utilized for in vitro prediction of Fa% of drugs that follow the transcellular passive transport route.

Immobilized phospholipid capillary electrophoresis for study of drug-membrane interactions and prediction of drug activity.

Immobilized phospholipid capillary electrophoresis (IPCE) was developed for studying the interactions between a set of nonsteroidal anti-inflammatory drugs (NSAIDs) and membrane and predicting the biological activity of NSAIDs. Supported vesicle layers and supported phospholipid bilayers were attached to the inner surface of a capillary wall simply by rinsing with liposome solutions. The liposomes, composed of soybean phosphatidylcholine (SPC) or SPC and different proportions of cholesterol (Ch), were small unilamellar vesicles prepared by sonication. The normalized capacity factor (K(IPCE)) was introduced into IPCE for evaluating drug-membrane interactions. Related theories and equations were derived to calculate K(IPCE) values from apparent migration time of a solute and electroosmotic flow. The strong relationships were observed between logK(IPCE) (SPC) values and logK(lw) values (the partition coefficients determined in free SPC-liposome partitioning system) (R=0.9855 and P<0.0001) or logK(ILC) values (the normalized capacity factors determined by immobilized POPC-liposome chromatography, POPC represents 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) (R=0.9875 and P<0.0001). In addition, logK(IPCE) (SPC/Ch 80:20%) values correlated well with the pIC50 (the minus logarithm of IC50) values for cyclooxygenase 2 determined on intact cells (R=0.959 and P<0.001). These results confirmed that IPCE, K(IPCE) value as evaluation index, can be effectually used for studying drug-membrane interactions and it has the potential to predict drug activity. Cholesterol-containing (20 mol%) liposomes may be more suitable to mimic real cell membrane.

[Chemical constituents from aerial parts of Plumbago zeylanica Linn].

OBJECTIVE: To investigate the chemical constituents of the aerial parts of Plumbago zeylanica Linn. METHODS: The constituents of the EtOAc-soluble portion in the 95% ethanol extract were isolated and purified by means of chromatography. Compounds were identified by their physical characteristics and spectral features. RESULTS: Nine compounds were isolated as plumbagin (I), isoshinanolone (II), plumbagic acid (III), beta-sitosterol (IV), 4-hydroxybenzaldehyde (V), trans-cinnamic acid (VI), vanillic acid (VII), 2, 5-dimethyl-7-hydroxychromone (VIII), indole-3-carboxaldehyde (IX). CONCLUSION: Compounds V, VII, VIII and IX were isolated for the first time from Plumbago Linn.

High-performance liquid chromatography of sulfonamides and quinolones on p-tert-butyl-calix[6]arene-bonded silica gel stationary phase.

The high-performance liquid chromatographic behavior of some sulfonamides and quinolones was studied on a p-tert-butyl-calix[6]arene-bonded silica gel stationary phase. The effect of mobile phase variables such as methanol content, ionic strength and pH on their chromatographic behavior was investigated. The retention behavior of sulfonamides on the stationary phase was compared with that on both Zorbax C(18)-bonded silica gel and gamma-(ethylenediamino)propyltriethoxylsilane-bonded silica gel (diamino-bonded phase). The retention mechanism of sulfonamides and quinolones on the stationary phase was also discussed. The results indicate that the stationary phase behaves as a reversed-phase packing and its separation selectivity is much better than that of not only Zorbax C(18) phase but also diamino-bonded phase. Some sulfonamides and quinolones were separated on the stationary phase, but the separation of sulfonamides is far more successful.