雷公藤内酯醇-溶菌酶偶联物的性能研究及体外评价

目的 研究肾靶向前体药物雷公藤内酯醇-溶菌酶偶联物的理化性质.方法 采用HPLC法考察偶联物在37℃不同pH缓冲液、大鼠血浆及大鼠肾脏溶酶体溶液中的稳定性及细胞毒性.结果 体外稳定性试验表明,偶联物在37℃的不同pH缓冲液及大鼠血浆中稳定,在大鼠肾脏溶酶体溶液中可降解.偶联物能降低肾近端小管的细胞毒性,减少脂多糖引导的NO产生.结论 雷公藤内酯醇-溶菌酶偶联物可提高雷公藤内酯醇的肾靶向性,并可降低不良反应.     

Triptolide alters barrier function in renal proximal tubular cells in rats

Alteration of the tight junction complex in renal epithelial cells can affect renal barrier function and perturb normal kidney homeostasis. The objective of the present study was to determine whether triptolide could affect tight junctions in the proximal tubule epithelial cells both in vivo and in vitro. Wistar rats were gavaged with triptolide at 0, 100, 200 or 400 μg/kg/day for 28 days. Pathologic examination of the kidney showed that triptolide primarily affected the proximal tubules. The nephrotoxicity of triptolide is morphologically characterized by the detachment of the proximal tubular epithelial cells from each other. Immunohistochemical analysis showed that there was marked alteration in the localization of Zonula Occludens 1 protein (ZO-1) in the proximal tubule epithelium. Additionally, the uptake of FITC-dextran, a marker of fluid phase endocytosis in the proximal tubule, was considerably lower in triptolide-treated animals than in normal rats. Supported by these results, we detected significant increases in blood urea nitrogen (BUN) but not of creatinine (Cr) in rats treated with triptolide, indicating damage to the proximal tubules. Furthermore, triptolide treatment caused an alteration of the tight junction complex, resulting in changes in paracellular permeability in NRK-52E cells in vitro. Taken together, these results suggest that triptolide induced renal toxicity in rats and that the mechanism of toxicity was related to the disruption of cell–cell junctions and alterations of the paracellular permeability in the proximal tubule.     

Targeting of Doxorubicin to the Urinary Bladder of the Rat Shows Increased Cytotoxicity in the Bladder Urine Combined With An Absence of Renal Toxicity

Targeting of anti-tumor drugs to the urinary bladder for the treatment of bladder carcinoma may be useful, since these agents generally have a low degree of urinary excretion and are highly toxic elsewhere in the body. The anti-tumor drug doxorubicin was coupled to the low-molecular weight protein lysozyme via the acid-sensitive cis -aconityl linker. All free amino groups of the lysozyme were used for drug attachment to achieve intact excretion of the doxorubicin-aconityl-lysozyme conjugate into the bladder. In the bladder, the cytotoxic drug should be regenerated through acidification of the urine. First, the doxorubicin-aconityl-lysozyme conjugate was tested in rats for its target specificity and general toxicity. Wistar rats were injected intravenously with 2 mg/kg free doxorubicin or 10 mg/kg lysozyme-conjugated doxorubicin. Total urinary excretion of doxorubicin was about 10 times higher if the drug was coupled to lysozyme (39 ± 3% versus 4.4 ± 0.4%) . Free doxorubicin had no detectable toxic effects on heart, liver and lung but caused severe renal damage (proteinuria, N -acetyl-glucosaminidase excretion and glomerulosclerosis). None of the rats injected with doxorubicin-lysozyme conjugate showed such renal toxicity. Second, we tested whether doxorubicin could be released from the conjugate in the bladder through acidification of the urine and if the released doxorubicin could still exert a cytotoxic effect. Doxorubicin-aconityl-lysozyme (2 mg/kg conjugated doxorubicin, i.v.) was administered in rats with acidified urine (pH 6.1 ± 0.1) and in rats with a high urinary pH (8.2 ± 0.4). Ten times more doxorubicin was released from the conjugate in the group with acidified urine (15 ± 7% versus 1.7 ± 0.1%) . In agreement with this, cytotoxicity was also higher in the low pH group (IC 50 of 255 ± 47 nM versus 684 ± 84 nM doxorubicin). In conclusion, a specific delivery of doxorubicin to the urinary bladder combined with a reduced toxicity of doxorubicin in the kidneys can be achieved by coupling this anti-tumor drug to the low-molecular weight protein lysozyme via an acid-labile linker. A release of cytotoxic doxorubicin in the urinary bladder can be achieved by acidification of the urine. This technology, after further optimization, may provide an interesting tool for the treatment of bladder carcinoma.     

Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of 1,4-naphthoquinone and menadione

We have previously demonstrated that administration of various benzoquinol-glutathione (GSH) conjugates to rats causes renal proximal tubular necrosis and the initial lesion appears to lie within that portion of the S3 segment within the outer stripe of the outer medulla (OSOM). The toxicity may be a consequence of oxidation of the quinol conjugate to the quinone followed by covalent binding to tissue macromolecules. We have therefore synthesized the GSH and N-acetylcysteine conjugates of 2-methyl-1,4-naphthoquinone (menadione) and 1,4-naphthoquinone. The resulting conjugates have certain similarities to the benzoquinol-GSH conjugates, but the main difference is that reaction with the thiol yields a conjugate which remains in the quinone form. 2-Methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone caused a dose-dependent (50-200 mumol/kg) necrosis of the proximal tubular epithelium. The lesion involved the terminal portion of the S2 segment and the S3 segment within the medullary ray. At the lower doses, that portion of the S3 segment in the outer stripe of the outer medulla displayed no evidence of necrosis. In contrast, 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) caused no apparent histological alterations to the kidney. 2-(Glutathion-S-yl)-1,4-naphthoquinone and 2,3-(diglutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) were relatively weak proximal tubular toxicants and the lesion involved the S3 segment at the junction of the medullary ray and the OSOM. A possible reason(s) for the striking difference in the toxicity of the N-acetylcysteine conjugate of menadione, as opposed to the lack of toxicity of the GSH conjugate of menadione, is discussed. The basis for the localization of the lesion caused by 2-methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone requires further study.     

Targeting of doxorubicin to the urinary bladder of the rat shows increased cytotoxicity in the bladder urine combined with an absence of renal toxicity

Targeting of anti-tumor drugs to the urinary bladder for the treatment of bladder carcinoma may be useful, since these agents generally have a low degree of urinary excretion and are highly toxic elsewhere in the body. The anti-tumor drug doxorubicin was coupled to the low-molecular weight protein lysozyme via the acid-sensitive cis-aconityl linker. All free amino groups of the lysozyme were used for drug attachment to achieve intact excretion of the doxorubicin-aconityl-lysozyme conjugate into the bladder. In the bladder, the cytotoxic drug should be regenerated through acidification of the urine. First, the doxorubicin-aconityl-lysozyme conjugate was tested in rats for its target specificity and general toxicity. Wistar rats were injected intravenously with 2 mg/kg free doxorubicin or 10 mg/kg lysozyme-conjugated doxorubicin. Total urinary excretion of doxorubicin was about 10 times higher if the drug was coupled to lysozyme (39 +/- 3% versus 4.4 +/- 0.4%). Free doxorubicin had no detectable toxic effects on heart, liver and lung but caused severe renal damage (proteinuria, N-acetylglucosaminidase excretion and glomerulosclerosis). None of the rats injected with doxorubicin-lysozyme conjugate showed such renal toxicity. Second, we tested whether doxorubicin could be released from the conjugate in the bladder through acidification of the urine and if the released doxorubicin could still exert a cytotoxic effect. Doxorubicin-aconityl-lysozyme (2 mg/kg conjugated doxorubicin, i.v.) was administered in rats with acidified urine (pH 6.1 +/- 0.1) and in rats with a high urinary pH (8.2 +/- 0.4). Ten times more doxorubicin was released from the conjugate in the group with acidified urine (15 +/- 7% versus 1.7 +/- 0.1%). In agreement with this, cytotoxicity was also higher in the low pH group (IC50 of 255 +/- 47 nM versus 684 +/- 84 nM doxorubicin). In conclusion, a specific delivery of doxorubicin to the urinary bladder combined with a reduced toxicity of doxorubicin in the kidneys can be achieved by coupling this anti-tumor drug to the low-molecular weight protein lysozyme via an acid-labile linker. A release of cytotoxic doxorubicin in the urinary bladder can be achieved by acidification of the urine. This technology, after further optimization, may provide an interesting tool for the treatment of bladder carcinoma.     

人参皂苷Rb_1与Rg_1对肾小管细胞缺氧复氧损伤模型的影响

目的:探讨人参皂苷的2种单体成分Rb1、Rg1在肾小管细胞缺氧复氧(HRO)损伤模型中的作用及机制。方法:将大鼠肾小管上皮细胞分为对照组、HRO组、Rb1+HRO组和Rg1+HRO组。除对照组外,其余各组给药同时缺氧复氧培养36h后分别检测肾小管细胞增殖活性、增殖细胞核抗原(PCNA)阳性表达情况、丙二醛(MDA)含量、乳酸脱氢酶(LDH)漏出量和超氧化物歧化酶(SOD)活性。结果:人参皂苷Rb1、Rg1可促进HRO性肾小管细胞增殖(P<0.01),增强细胞中PCNA阳性表达(P<0.05),减少肾小管细胞MDA生成及LDH释放,增强SOD活性。结论:人参皂苷Rb1和Rg1对HRO性肾小管细胞损伤模型具有保护作用。     

Nephrotoxicity of 4-aminophenol glutathione conjugate.

4-Aminophenol (p-aminophenol, PAP) causes selective necrosis to the pars recta of the proximal tubule in Fischer 344 rats. The basis for this selective toxicity is not known, but PAP can undergo oxidation in a variety of systems to form the 4-aminophenoxy free radical. Oxidation or disproportionation of this radical will form 1,4-benzoquinoneimine which can covalently bind to tissue macromolecules. Recent studies have shown that certain benzoquinol-glutathione conjugates can cause renal necrosis in rats. We have synthesized a putative glutathione conjugate of PAP. The effect on the kidney of this conjugate and the sulphate and N-acetyl conjugates, known metabolites of PAP, have been examined in Fischer 344 rats. 4-Amino-3-S-glutathionylphenol produced a dose-dependent (92-920 mumol kg-1) necrosis of the proximal tubular epithelium and altered renal excretory function. The lesion at the low dose was restricted to the pars recta of the proximal tubule in the medullary rays, while at the higher doses it affected the pars recta region of all nephrons. In contrast, PAP-O-sulphate and N-acetyl-4-aminophenol (paracetamol) caused no histological or functional alteration to the kidney at 920 mumol kg-1. The renal necrosis produced by 4-amino-3-S-glutathionylphenol was very similar to that produced by PAP (367-920 mumol kg-1), both functionally and histologically, except that smaller doses of the glutathione conjugate were required. These studies indicate that glutathione conjugation of PAP generates a metabolite that is more toxic to the kidney than the parent compound. A possible mechanism of toxicity (analogous to that reported for glutathione conjugates of certain quinones) involving oxidation to form a 1,4-benzoquinoneimine thioether that could redox cycle is discussed.     

Renal targeting of kinase inhibitors

Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.     

Preparation and characterization of a biodegradable drug targeting system for anticancer drug delivery: Microsphere-antibody conjugate

Targeted delivery of anticancer drugs is one of the most actively pursued goals in anticancer chemotherapy. A major disadvantage of anticancer drugs is their lack of selectivity for tumour tissue, which causes severe side effects and results in low cure rates. Any strategy by which a cytotoxic drug is targeted to the tumour, thus increasing the therapeutic index of the drug, is a way of improving cancer chemotherapy and minimizing systematic toxicity. This study covers the preparation of the gelatin microsphere (GM)-anti-bovine serum albumin (anti-BSA) conjugate for the development of a drug targeting approach for anticancer drug delivery. Microspheres of 5% (w/v) gelatin content were prepared by crosslinking with glutaraldehyde (GTA) at 0.05 and 0.50% (v/v) concentration. Microspheres were in the size range of 71–141 μm. The suitability of these microspheres as drug carriers for anticancer drug delivery was investigated in vitro by studying the release profiles of loaded methotrexate (MTX) and 5-fluorouracil (5-FU) and the cytotoxicities on cancer cell lines. The in vitro MTX release profiles (～22–46% released in 24 h depending on the amount of GTA used) were much slower compared to 5-FU (～42–91% released in 24 h). Both drugs demonstrated an initial fast release, which was followed by gradual, sustained drug release. The MTT cytotoxicity test results of GMs loaded with 5-FU and MTX showed ～54–70% and ～52–67% cytotoxicities in 4 days. In general, incorporation of MTX and 5-FU in microspheres enhanced the cytotoxic effect in a more prolonged manner compared to the free drugs. Gelatin micospheres were chemically conjugated to anti-BSA and the antigen–antibody activities were studied by immunofluorescence. Results indicated ～80% binding with conjugated anti-BSA and BSA-FITC. Based on their low cytotoxicity and the high antigen binding efficiencies, anti-BSA conjugated gelatin microspheres could be suitable targeted drug carrier systems for selective and long-term delivery of anticancer drugs to a specific body compartment (i.e. bladder cancer).     

Synthesis and characterization of the tumor targeting mitoxantrone-insulin conjugate

Anticancer drugs have serious side effects arising from their poor malignant cells selectivity. Since insulin receptors highly express on the cytomembrane of some kind of tumor cells, using insulin as the vector was expected to reduce serious side effects of the drugs. The objective of this study was to evaluate the tumor targeting effect of the newly synthesized mitoxantrone-insulin conjugate (MIT-INS) with the drug loading of 11.68%. In vitro stability trials showed MIT-INS were stable in buffers with different pH (2-8) at 37 degrees C within 120 h (less than 3% of free MIT released), and were also stable in mouse plasma within 48 h (less than 1% of free MIT released). In vivo study on tumor-bearing mice showed that, compared with MIT [75.92 microg x h/g of the area under the concentration-time curve (AUC) and 86.85 h of mean residence time (MRT)], the conjugates had better tumor-targeting efficiency with enhanced tumor AUC of 126.53 microg x h/g and MTR of 151.95 h. The conjugate had much lower toxicity to most other tissues with targeting indexes (TIC) no larger than 0.3 besides good tumor targeting efficiency with TIC of 1.67. The results suggest the feasibility to promote the curative effect in cancer chemotherapy by using insulin as the vector of anti-cancer drugs.     

Cadmium toxicity is caused by accumulation of p53 through the down-regulation of Ube2d family genes in vitro and in vivo

Cadmium (Cd) causes renal dysfunction with damage to kidney proximal tubule cells; however, the precise mechanisms of the toxicity remain unclear. Previously, we found that the expression of Ube2d4 gene, which is a member of the ubiquitin-conjugating enzyme Ube2d family, is suppressed by Cd in NRK-52E rat renal tubular epithelial cells. To investigate the mechanisms of Cd-induced renal toxicity, we examined the effects of Cd on the ubiquitin-proteasome system, particularly the expression and function of Ube2d family members in the NRK-52E cells and mice. Cd markedly decreased the expression of Ube2d1, Ube2d2, Ube2d3 and Ube2d4 prior to the appearance of cytotoxicity in the NRK-52E cells. Cd also dramatically increased p53 protein levels in the cells, without stimulation of p53 gene expression or inhibition of proteasome activity. In addition, Cd induced phosphorylation of p53 and caused apoptosis in the NRK-52E cells. In vivo, we examined the effect of orally administrated Cd for 12 months on the expression of Ube2d genes and accumulation of p53 in the mouse kidney. Chronic Cd exposure also caused suppression of Ube2d genes expression and accumulation of p53. Cd did not induce severe kidney injury, but caused apoptosis in the renal tubules. These results suggest that the Cd-induced accumulation of p53 may be due to inhibition of p53 degradation through the down-regulation of Ube2d family genes, and that Cd induces p53-dependent apoptosis in renal tubular cells. Moreover, Ube2d family members may be one of the critical targets of renal toxicity caused by Cd.     

不同分子量葡聚糖-地塞米松连接物的体外靶向释药特性不同分子量葡聚糖-地塞米松连接物的体外靶向释药特性

目的筛选具有结肠靶向性的葡聚糖-地塞米松连接物,探讨葡聚糖分子量对连接物体外释药特性的影响。方法将不同分子质量葡聚糖-地塞米松连接物与大鼠胃肠道不同部位内容物稀释液一起孵育,采用反相高效液相色谱法检测地塞米松及地塞米松琥珀酸单酯的释放情况。结果在160 min孵育过程中,胃内容物中未检测到释放的地塞米松及地塞米松琥珀酸单酯;DexD26和DexD50在结肠及盲肠内容物中释放出地塞米松(包括地塞米松琥珀酸单酯)的总量分别是其在小肠近端及小肠远端内容物中释放总量的4.0和3.6倍;DexD2和DexD7.6在结肠及盲肠内容物中释放出地塞米松(包括地塞米松琥珀酸单酯)的总量分别是其在小肠近端及小肠远端内容物中释放总量 的2.0和1.9倍。结论葡聚糖分子质量对连接物的体外释药特性有明显影响,大分子质量葡聚糖-地塞米松连接物具有较大的结肠定位释放潜力。     

Characterization of in vivo disulfide-reduction mediated drug release in mouse kidneys

Due to the overexpression of a folate receptor (FR) on many malignant cells, folate-targeted drugs have been developed to improve the cancer specificity of chemotherapeutic agents. Therapeutic index is further enhanced with the use of self-immolative linkers that efficiently release the attached drug upon cellular internalization of the folate-drug conjugate. Because FR is also abundant in normal kidney proximal tubule (PT) cells, we sought to examine in real time the trafficking and release of folate-targeted drugs in the kidney in vivo. Thus, we conducted two-photon kidney imaging studies in mice utilizing a F?rster resonance energy transfer (FRET) based folate conjugate that undergoes a color shift from red to green upon reduction of the disulfide bond linking folate to a surrogate drug molecule. Following infusion via intravenous injection, folate-FRET reached the kidney in its intact unreduced form. The folate-FRET conjugate was then filtered into the lumen of PT, where it was efficiently captured by FR. As FR transcytosed across PT, some disulfide reduction occurred, with reduced folate-FRET detectable in PT vesicles 30 min postinjection. Prolonged monitoring of folate-FRET in mice showed modest progression of reduction in PT cells over time. Moreover, inhibition of FR trafficking in PT cells by colchicine did not significantly affect the rate or extent of folate-FRET reduction. Finally, the lack of cytosolic accumulation of released drug surrogate in the PT suggests that drug release via disulfide bond reduction should cause little kidney toxicity.     

Dual Effects of Bilirubin on the Proliferation of Rat Renal NRK52E Cells and ITS Association with Gap Junctions

Objective:

The effect of bilirubin on renal pathophysiology is controversial. This study aimed to observe the effects of bilirubin on the proliferation of normal rat renal tubular epithelial cell line (NRK52E) and its potential interplay with gap junction function.

Methods:

Cultured NRK52E cells, seeded respectively at high- or low- densities, were treated with varying concentrations of bilirubin for 24 hours. Cell injury was assessed by measuring cell viability and proliferation, and gap junction function was assessed by Parachute dye-coupling assay. Connexin 43 protein was assessed by Western blotting.

Results:

At doses from 17.1 to 513μmol/L, bilirubin dose-dependently enhanced cell viability and colony-formation rates when cells were seeded at either high- or low- densities (all p<0.05 vs. solvent group) accompanied with enhanced intercellular fluorescence transmission and increased Cx43 protein expression in high-density cells. However, the above effects of BR were gradually reversed when its concentration increased from 684 to 1026μmol/L. In high-density cells, gap junction inhibitor 12-O-tetradecanoylphorbol 13-acetate attenuated bilirubin-induced enhancement of colony-formation and fluorescence transmission. However, in the presence of high concentration bilirubin (1026μmol/L), activation of gap junction with retinoid acid decreased colony-formation rates.

Conclusion:

Bilirubin can confer biphasic effects on renal NRK52E cell proliferation potentially by differentially affecting gap junction functions.     

Low Molecular Weight Proteins as Carriers for Renal Drug Targeting: Naproxen–Lysozyme

Low molecular weight proteins (LMWPs), such as lysozyme, may be suitable carriers to target drugs to the kidney. In this study the antiinflammatory drug naproxen was covalently bound to lysozyme (1:1). Pharmacokinetics of the conjugate, naproxen–lysozyme (nap-LYSO), were compared to that of an equimolar mixture of uncoupled naproxen with lysozyme in freely moving rats. Similar plasma kinetics and organ distribution for native lysozyme and the drug conjugate were observed (Cl_p = 1.2 and 1.1 ml/min; = 85 and 75 min, respectively). In case of the uncoupled naproxen–lysozyme mixture, a monoexponential plasma disappearance of naproxen with a of 2.8 hr was observed, coinciding with urinary excretion of naproxen metabolites (mainly 6-desmethylnaproxen sulfate; 6-DMN-S) between 2 and 8 hr after injection. Urinary recovery of total metabolites was 59% of the naproxen dose. In contrast, after injection of covalently bound naproxen, plasma levels of the parent drug were below the detection level, whereas naproxen was recovered as 6-DMN-S in urine over a period from 4 to 30 hr. However, only 8% of the administered dose was recovered as 6-DMN-S in urine, whereas 50% of the dose was recovered as naproxen metabolites in feces. Incubation experiments using purified renal tubular lysosomal lysates revealed that naproxen–lysozyme degradation ultimately results in a stable naproxen amino acid catabolite, naproxen–lysine (nap-lys). Hepatic uptake and biliary excretion of this catabolyte were demonstrated in isolated perfused rat livers. Further, an equipotent pharmacological activity relative to parent naproxen was observed. We conclude that LMWPs such as lysozyme are indeed suitable carriers for site-specific delivery of drugs to the kidney. Although naproxen covalently bound to lysozyme did not release the parent drug, it did result in renal release of a stable and active catabolite, naproxen–lysine.     

Angiotensin AT1 receptor activation mediates high glucose‐induced epithelial–mesenchymal transition in renal proximal tubular cells

1. Renal tubular epithelial cells can undergo epithelial to mesenchymal transition (EMT) under hyperglycaemic conditions, which is associated with renal interstitial fibrosis. Activation of the renin–angiotensin system (RAS) is involved in diabetic nephropathy. The present study investigated the positive role of angiotensin AT₁ receptors in high glucose‐induced EMT in cultured tubular epithelial cells. 2. A rat kidney proximal tubular epithelial cell line (NRK‐52E) was used in the present study. Levels of EMT makers, namely E‐cadherin and vimentin, were estimated using fluorescence immunocytochemistry, mRNA levels of angiotensinogen (AGT), angiotensin‐converting enzyme (ACE) and AT₁ receptors were determined by real‐time polymerase chain reaction, protein levels of E‐cadherin, vimentin, fibronectin, matrix metallopeptidase (MMP)‐9 and phosphorylated extracellular signal‐regulated kinase (ERK) 1/2 were analysed by western blotting and the concentrations of angiotensin (Ang) II and transforming growth factor (TGF)‐β1 in the culture medium were determined by enzyme immunoassay and ELISA. 3. High glucose (30 mmol/L) induced EMT and increased the synthesis of fibronectin and MMP‐9. Furthermore, high glucose increased AGT, ACE and AT₁ receptor mRNA levels, as well as AngII and TGF‐β1 concentrations in the culture medium and ERK1/2 phosphorylation. Pretreatment of cells for 15 min with the AT₁ receptor antagonist losartan (10^?5 mol/L) attenuated high glucose‐induced increases in TGF‐β1 and ERK1/2 phosphorylation and reduced EMT, as well as the consequent synthesis of fibronectin and MMP‐9. 4. The results of the present study suggest that the activated local RAS mediates high glucose‐induced EMT. By activating AT₁ receptors and stimulating TGF‐β1 synthesis, the elevated local RAS participates in high glucose‐induced EMT and increased extracellular matrix secretion.     

Influence of end groups on in vitro release and biological activity of lysozyme from a phase-sensitive smart polymer-based in situ gel forming controlled release drug delivery system

Phase-sensitive in situ gel forming controlled release formulations of lysozyme were prepared using poly lactic acid (PLA) and/or poly glycolic acid (PGA) based polymers differing in end groups in addition to composition, and a solvent system consisting of various ratios of benzyl benzoate (BB) and benzyl alcohol (BA). The amount of lysozyme in the released samples was determined by measuring absorbance at 280 nm using suitable controls to nullify the effect of absorption of formulation degradation products. Biological activity of lysozyme was studied by an enzyme activity assay using Micrococcus lysodeikticus as substrate. Polymers bearing carboxylic acid end group were not soluble in 100% BB but polymers having ester end groups were soluble up to 27% (w/v) except polymer 4. A biphasic release profile consisting of slower first phase followed by faster second phase was observed. Formulations prepared from polymer with carboxylic acid groups showed significantly (p<0.05) lower burst release (4%) than those containing ester end groups (20-30%). However, formulations consisting of polymer with carboxylic acid end groups showed significantly (p<0.05) faster release rate of incorporated lysozyme, although the total amount released was less in comparison to the total amount released from formulations prepared using polymers containing ester end groups. The mean percentage specific enzyme activity (MPSEA) data were supported by the release profiles. In conclusion, polymer end groups may influence the release profiles of a protein from an in situ gel depot forming controlled release formulations.     

新穿膜融合蛋白His-T1-绿色荧光蛋白的跨膜效率及其对细胞存活的影响

目的研究穿膜融合蛋白His-T1-绿色荧光蛋白(GFP)的跨膜效率及其对细胞存活的影响。方法以浓度为500mg·L-1的His-T1-GFP与人鼻咽癌CNE2或大鼠肾小管上皮NRK52E细胞孵育6h,应用荧光显微镜观察His-T1-GFP跨膜进入细胞的情况。应用多功能酶标仪检测细胞荧光强度,研究His-T1-GFP跨膜的动力学因素:以浓度为500mg·L-1的His-T1-GFP与CNE2或NRK52E细胞孵育10min至24h,观察孵育时间对穿膜作用的影响;以浓度为25mg·L-1至1.0g·L-1的His-T1-GFP与两种细胞孵育6h,观察蛋白浓度对跨膜效率的影响;以浓度为500mg·L-1的His-T1-GFP与两种细胞分别在4℃和37℃的条件下孵育6h,观察温度对蛋白跨膜效率的影响。用细胞乳酸脱氢酶(LDH)试剂盒和MTT法来评价5.0g·L-1浓度的His-T1-GFP对细胞存活的影响。结果在一定浓度范围内,His-T1-GFP能够有效穿透CNE2和NRK52E细胞膜,且对NRK52E细胞的跨膜效率明显高于His-TAT-GFP。His-T1-GFP在10min内就能有效跨膜进入细胞,并且在6h内进入细胞的量与时间成正相关。在一定浓度范围(25mg·L-1~1.0g·L-1)内,该蛋白进入细胞的量与自身浓度成正相关,而在4℃时该蛋白仍具有跨膜能力。当其终浓度高达5.0g·L-1时,对CNE2和NRK52E两种细胞几乎无毒性作用。结论His-T1-GFP蛋白是一种跨膜效率高且低毒的穿膜融合蛋白。