Effect and molecular mechanism research of Astragalus membranaceus on inhibiting intestinal absorption of six alkaloids of Aconitum carmichaelii in spleen deficiency rats / 中草药·英文版

Objective: To investigate the effect and the mechanism of Astragalus membranaceus (Huangqi in Chinese, HQ) extract on the intestinal absorption of six alkaloids of Aconitum carmichaelii (Fuzi in Chinese, FZ) in rats with spleen deficiency and provide novel insights into the application of HQ on modulating intestinal barrier. Methods: Four-week-old male Sprague-Dawley rats were fed with Xiaochengqi Decoction to induce the spleen deficiency model for 40 d. Single-pass intestinal perfusion model were used to study the effects of HQ extract on the absorption of alkaloids. Protein expression and mRNA levels of MRP2 and BCRP and tight junction proteins (TJ, including Claudin-1, Occludin and ZO-1) were measured using Western blot and real-time PCR, respectively. The location and expression of TJ protein was also investigated by the immunofluorescence method. Results: Compared with the normal group, the protein expression of MRP2, BCRP and TJ proteins in the model group were significantly down-regulated. After oral administration of HQ, the alkaloid absorption in intestinal villi was inhibited, MRP2, BCRP and TJ proteins were up-regulated, the green fluorescence staining of Claudin-1, Occludin, and ZO-1 was enhanced, and a thick layer of mucus was deposited on the surface of the epithelium of the intestinal cavity. Conclusion: HQ as an intestinal barrier modulator improves the physiological changes of the intestinal environment of spleen deficiency to reduce the absorption of toxic components, leading to a decrease in the absorption of drug-like molecules.

Research on Therapeutic Mechanism of Canhuang Tablets on Jaundiced Rats Induced by ANIT / 中国实验方剂学杂志

Objective:To explore the therapeutic mechanism of Canhuang tablets on the mRNA and protein expression of farnesoid X receptor (FXR), uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) and multidrug resistance associated protein 2 (MRP2) in the liver of jaundiced rats induced by α-naphthalene isothiocyanate (ANIT). Method:The rats were divided into normal group, model group, Canhuang tablets (CHP) group and ursodeoxycholic acid tablets (UDCA) group. The jaundice model was reproduced by ANIT. After the intervention of the corresponding drugs, the contents of total bilirubin (TBIL), total bile acid (TBA), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum and the liver histopathology were examined to evaluate the therapeutic effect of CHP. The relative mRNA and protein expressions of FXR, UGT1A1 and MRP2 in rat liver tissues were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Result:CHP can significantly reduce the increase of TBIL, TBA, ALT, AST and ALP caused by ANIT in rat serum, and inhibit the liver pathological changes, which showed that the removing jaundice effect of CHP was better than UDCA. Compared with the normal group, ANIT significantly inhibited the mRNA levels of FXR, UGT1A1 and MRP2 in rat liver tissues after modeling (P<0.01). Compared with the model group, CHP and UDCA significantly increased the mRNA levels of target genes of each protein after intervention (P<0.01), and CHP was superior to UDCA in improving the mRNA level of bilirubin metabolizing enzyme UGT1A1 (P<0.01). In the aspect of affecting protein expression, compared with the normal group, ANIT modeling significantly increased the expression of FXR in rats (P<0.05). CHP intervention showed a tendency to promote the expression of FXR, while UDCA did not, but there was no significant difference between them. In the aspects of promoting bilirubin metabolism and bile excretion, the expressions of UGT1A1 and MRP2 were significantly decreased by ANIT modeling (P<0.01), while the expressions of UGT1A1 and MRP2 proteins were significantly increased after treatment of CHP (P<0.01). CHP was superior to UDCA in increasing the expression of bilirubin and bile acid efflux protein MRP2 (P<0.01). Conclusion:The jaundice abating mechanism of CHP is related to activating FXR mRNA expression in liver, promoting the mRNA and protein expression of bilirubin metabolizing enzyme UGT1A1 and bile acid transporter MRP2, improving liver metabolism of free bilirubin and promoting bile acid excretion from the liver, and alleviating cholestatic liver injury.

Maternal Near Miss Cases in Medical College Ambikapur Running in Tribal Zone of Chhattisgarh: A Retrospective Study

MMR being an important index for evaluation of obstetric care of that area has a little information regarding actions to be taken to negotiate the maternal health issues of that area. Hence more number of cases who were moribund and critical but fortunately escaped death grouped as near miss was being studied to find out the shadowed causes of maternal mortality. Methods: All 312 near miss cases women were evaluated out of total 6040 admissions in obstetric ward of this institute during a period of one year from April 2017 to March 2018 to know the steps to be taken for improvement of maternal health in tribal and low resource newly started medical college. Result: Hypertensive disorders of pregnancy are the main culprit of maternal mortality whereas hemorrhage and its aftereffects are proved to be the most important cause of near miss. Incomplete abortion is the most common cause of hemorrhage and over the counter sell and misuse of Mifegest is found mainly responsible for incomplete abortion. Conclusion: To improve the obstetric care we have to educate the public for proper antenatal checkup. It will help health workers to identify the high risk pregnancies and their timely management. Apart from public awareness program there is necessity of well-equipped government health set ups where the poor tribal and also the other needy ones can report easily with all faith and confidence. Scarcity of skilled staff is also a subject to be noticed.

Potential mechanism of cholagogic effect about Gardenia Jasminoides Ellis (Zhizi)-mediated increase of bile acids urinary excretion in normal rats / 中草药·英文版

Objectives: Gardenia Jasminoides Ellis (Zhizi), belonging to Rubiaceae family, has been traditionally used for treatment cholestasis and jaundice for centuries in Asian countries. In the theory of Traditional Chinese Medicines, Zhizi could dispel dampness and heat via the urine to execute its choleretic effects. However, the potential molecular mechanism has been still poorly clarified. Here, we investigated the effect of different dose of Zhizi aqueous extract powder (0.3 g/kg/d and 0.9 g/kg/d) on urinary excretion of bile acids (BAs), and defined the potential mechanism via renal BAs efflux transporters Mrp2 and Mrp4 in normal rats. Methods: Male Wistar rats were orally administrated with 0.3 or 0.9 g/kg/d dose of Zhizi aqueous extract powder for 2 weeks, then body weight, serum aminotransferase, total BAs concentrations in liver, bile, serum, kidney and urine, 1 h bile flow, 12-h urinary volume, biliary and urinary excretion amount of total BAs as well as protein expression of major renal BAs efflux transporter Mrp2 and Mrp4, were all evaluated. Results: Zhizi especially the high dose of Zhizi aqueous extract powder could reduce hepatic total BAs concentration. Additionally, bile flow and biliary excretion had no significant difference, but the remarkable increasing urinary excretion of BAs and 2 to 3 folds up-regulated renal Mrp2 expression were observed after administrated with Zhizi as compared with the control group. Conclusion: The findings indicate that Zhizi reduces hepatic total BAs level by increasing urinary excretion rather than the biliary excretion of BAs, which, in turn ascribed to elevated protein expression of Mrp2 at apical membrane of renal tubular epithelial cells.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells / 中国天然药物

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 μmol·L. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

A novel chemo-resistant gene MSX2 discovered by establishment of two pancreatic cancer drug resistant cell lines JF305/CDDP and PANC-1/GEM / 中华肿瘤杂志

Objective@#To explore new multidrug resistant genes of pancreatic cancer by establishment and characterization of chemo-resistant cell lines.@*Methods@#The cisplatin-resistant cell line JF305/CDDP and the gemcitabine-resistant cell line PANC-1/GEM were induced by high-dose intermittent treatment. CCK-8 assay was used to detect the 50% inhibiting concentration (IC50), drug resistance index (R), cross-resistance, and growth difference of different cells. The changes of cell cycle and migration ability of drug-resistant cells were determined by flow cytometry and transwell assay, respectively. And then real-time fluorescence quantitative PCR was used to detect the expression of multidrug resistance-related genes.@*Results@#The drug resistance indexes of JF305/CDDP and PANC-1/GEM were 15.3 and 27.31, respectively, and there was cross-resistance. Compared with the parental cells, the proliferation rate of JF305/CDDP was decreased by 40% on the fourth day (P<0.05); the proportion of S phase was decreased from (45±2)% to (30±2)% (P<0.05), and the migration ability was enhanced from (32 ±1) cells per field to (158±5) cells per field (P<0.01). The expression of multidrug resistance-related genes MRP2, MDR1, LRP and MSX2 was increased in JF305/CDDP cells (P<0.05). Knockdown of MSX2 in JF305 cells reduced the expression of MRP2, whereas overexpression of MSX2 in PANC-1 cells upregulated MRP2 level (P<0.05).@*Conclusions@#Two stable multidrug resistant cell lines of pancreatic cancer, JF305/CDDP and PANC-1/GEM, were successfully established. MSX2 might be a new drug resistance related gene in pancreatic cancer cells by up-regulation of MRP2 expression.

The Protective effect of oleanolic acid on acute cholestatic liver injury in rats / 中华急诊医学杂志

Objective The protective effect of oleanolic acid on acute cholestatic liver injury in rats. Methods Thirty male SD rats were randomly divided into 3 groups ( n= 10 per group): sham group, bile duct ligated (BDL) group, and bile duct ligated with oleanolic acid (BDL+OA ) group. After 7 days, liver samples in all rats were collected. Expressions of bile acids pump and nuclear receptors at mRNA and protein levels were detected by RT- qPCR and Western blotting. Results At mRNA level, the expression of Mrp4 and Oatp1 expression in the BDL and BDL+OA groups were increased as compared with that in the sham group. The expression of Mrp4 increased 1.8 times in the BDL group and increased 2.3 times in the BDL+OA group (P<0.05), but the expression of Oatp1 was not statistically significant; AhR was increased 1.7-fold in the BDL group and 2.8 times in the BDL+OA group, Nrf2 was increased 1.5-fold in the BDL group and 2.1 times in the BDL+OA group with statistically significant difference. At the protein level, in the BDL group, Mrp4 expression increased 1.3 times, Oatp1 expression increased 1.5 times, AhR expression increased 1.3 times, Nrf2 expression increased 1.4 fold; in the BDL+OA group, Mrp4 expression increased 1.8 fold, AhR expression increased 1.9 fold, with statistical significance between the two groups. Oatp1 expression increased 1.4-fold in the BDL+OA group as compared with the BDL group showing no statistical significance. Conclusions Oleanolic acid stimulates the hepatic expression of bile acids pump Mrp4 associated with the activation of nuclear receptors AhR and Nrf2 in acute bile ductligated rats.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells / 中国天然药物

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 μmol·L. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

Research of Xiaoyan Decoction on multidrug resistance of lung adenocarcinoma A549/DDP cell with cisplatin resistance in human gene / 中草药

Objective: To study the Xiaoyan Decoction serum influence the expression level of lung adenocarcinoma that cisplatin-resistent of A549/DDP cell multidrug resistance-associated protein 1 (MRP1), lung resistance-related protein (LRP), MRP mRNA, and LRP mRNA, to find out Xiaoyan Decoction's target to the chemotherapy resistance, and to lay foundation for the clinical of reversing lung cancer chemotherapy resistance. Methods: The A549 nude mice hypodermic tumor model was given an equal amount of saline, 2 mg/kg of cisplatin, low and high doses of Xiaoyan Decoction (20, 40 g/kg), and then the serum was gain. The cisplatin resistance of human lung adenocarcinoma A549/DDP cell line was chosen as acquired drug-resistance model, Xiaoyan Decoction on MDR of A549/DDP cell gene expression product of multidrug resistance-associated protein 1, lung resistance-related protein and its effect on mRNA expression level was detected by Western blotting and RT-PCR technique. Results: Compared with the control group, the expression of MRP1 and LRP protein decreased gradually (P<0.05) as the serum drug concentration of Xiaoyan Decoction increased. The level of LRP mRNA and MRP1 mRNA in low and high doses of Xiaoyan Decoction group also decreased, and much more obvious inhibition of gene expression was observed as the increasing of drug concentration. Conclusion: Different serum drug concentration of Xiaoyan Decoction can differently control the expression of MRP1, LRP protein, MRP1 mRNA, and LRP mRNA, with the increasing of serum drug concentration, the decreasing of expression level, namely positive-correlated to the dose, which indicates that Xiaoyan Decoction Reversal of the lung cancer drug resistance the might related to the MRP1 and LRP.

Effect of arecoline hydrobromide on mRNA expression of hepatic and renal transporters in vivo / 中草药

Objective: To explore the effect of arecoline hydrobromide (AH) on the expression of rat hepatic and renal transporters. Methods: The effect of AH on the mRNA expression of 13 hepatic and renal transporters was studied after orally giver AH (0.8, 4, and 20 mg/kg/d) to rats for 21 d. Results: The results from the real-time PCR indicated that, AH treatment at low dose significantly decreased the mRNA levels of hepatic MRP2 and MDR1A, while significantly increased renal MRP5 mRNA level. On the other hand, AH treatment at high dose significantly inhibited the mRNA expression of hepatic OCT2, OAT2, OCTN2, OATP1A1, OATP1A4, OATP2B1, MRP2, and MDR1A, as well as renal MRP2, BCRP, and MDR1A. However, the mRNA expression of renal OCTN2, OATP1A1, OATP1A4, and MRP5 were significantly up-regulated following the treatment of high dose of AH. And the AH-induced effect on the above transporters was dose dependent in some extent. Conclusion: Due to the drug interaction caused by the alteration in expression and function of hepatic and renal transporters, it is suggested that the betel nut addicts should be paid more attention in case of adverse drug interactions.

Effects of magnesium isoglycyrrhizinate on the expressions of UGT1A, MRP2 protein and mRNA in L-02 cells damaged by triptolide / 天津医药

Objective To observe the effect of magnesium isoglycyrrhizinate on the expressions of UGT1A, MRP2 protein and mRNA of L-02 cells damaged by triptolide, and to investigate hepatoprotective mechanism of magnesium isoglycyrrhizinate in terms of drug metabolism. Methods L-02 cells were divided into 4 groups:normal group, triptolide group, magnesium isoglycyrrhizinate group and rifampicin group. Magnesium isoglycyrrhizinate group and rifampicin group were pretreated by magnesium isoglycyrrhizinate and rifampicin for 24 h and the remaining two groups added medium. Triptolide were added for 18 h except normal group. Cell survival rate was tested by MTT. The expression levels of UGT1A, MRP2 protein and mRNA were detected by Western blot assay and RT-PCR. Results Compared with triptolide group, cell survival rate was significantly higher in magnesium isoglycyrrhizinate group (P<0.05). Meanwhile, the expression levels of UGT1A, MRP2 protein and mRNA were significantly lower in triptolide group compared with those of control group (P<0.05). The expression levels of UGT1A, MRP2 protein and mRNA were significantly up-regulated in magnesium isoglycyrrhizinate pretreatment group than those of triptolide group (P<0.05). The UGT1A protein and mRNA expressions were significantly decreased in rifampicin pretreatment group than those of magnesium isoglycyrrhizinate group ( P<0.05), but there were no significant differences in MRP2 protein and mRNA expressions between the two groups. Conclusion Magnesium isoglycyrrhizinate shows protective effects on triptolide induced L-02 cell injury, which may be involved with the activation of UGT1A and MRP2.

Twist induces drug resistance in human lung cell line A549 by up-regulating MRP3 / 实用医学杂志

Objective To investigate the molecular mechanism of Twist induced-drug resistance in human lung cell line A549 cells.Methods The sensibility of A549 cells over-expressed Twist to cisplatin was measured by CCK8 assay. The expressions of Twist,MRP1,MRP2 and MRP3 were detected by RT-PCR. Western blotting was used to detect the expressions of Twist and MRP3 in A549 cells over-expressed Twist.The correlation between Twist and MRP3 expressions in 30 cases of lung cancer tissues was detected with immunohistochemistry. Results The viability of A549 cells over-expressed Twist was significant better than the control group under cispla-tin(32,64,128 μmol/L)treatment(0.79 ± 0.039,0.63 ± 0.048,0.46 ± 0.039 vs.0.53 ± 0.039,0.41 ± 0.043, 0.27 ± 0.063,respectively). After knockdown of MRP3 expression,it reversed drug resistance induced by Twist. Clinically,the expressions of Twist and MRP3 in lung cancer tissues were significantly relevant(P < 0.05). Conclusions Twist induces the drug resistance of human lung cell line A549 to cisplatin via up-regulating the expression of MRP3.

A Novel Decrease of Mrp3 Protein in Liver of β-Thalassemic Mouse.

Background: Hepatocytes have a fundamental system of efflux proteins that protect cells from toxic insults. Unconjugated bilirubin at higher concentration is toxic to cells and its intracellular accumulation is limited by the induction of efflux proteins such as Mrp3. In vivo studies showed an induction of hepatic Mrp3 expression in response to non-hemolytic hyperbilirubinemia as a compensatory mechanism to reduce UCB toxicity. Study Design: In the present study, we analyzed the hepatic Mrp3 expression profile during hemolytic hyperbilirubinemia. We used β-thalassemic mouse and WT rodents treated with phenylhydrazine as an animal model of chronic and acute hemolysis, respectively. Results: Unexpectedly, Mrp3 protein was 75% down-regulated in β-thalassemic mouse although Mrp3 mRNA was normal. Mrp3 mRNA was significantly induced in PHZ treated animals while again; Mrp3 protein was 60% down-regulated. Conclusion: For the first time we observed a clear down-regulation for hepatic Mrp3 protein that linked to hemolysis, not to bilirubin. We hypothesize that a similar decrease for hepatic Mrp3 proteins is occur in hemolytic patients such as β-thalassemia.

Studies on effects and mechanisms of P-gp and MRP1 inhibitor on transportation of gastrodin / 中草药

Objective: To study the effects and mechanisms of p-glycoprotein (P-gp) inhibitor and MRP1 inhibitor on the transportation of gastrodin (GAS). Methods: Cell toxicity of GAS was detected by MTT assay, molecular Docking was employed to predicted binding mode and effect ability of GAS with P-gp and MRP1. MDCK-MDR1 cell model was employed to study the influences of Ver, a P-gp inhibitor, and Probenecid, a MRP1 inhibitor, on the transportation of GAS. Results: There was no cell cytotoxicity of GAS between the concentration of 100 to 1000 μg/mL. There was hydrogen-bond and hydrophobic interaction between P-gp and GAS, and hydrogen-bond, hydrophobic interaction and electrostatic-interaction between MRP1 and GAS. LibDock Score indicated that both P-gp-verapamil (Ver) and MRP1-Probenecid were more stable than P-gp-GAS and MRP1-GAS. The Papp of GAS BL→AP was greater than that of AP→BL, Efflux ratio (ER) of GAS was about 1.5, which indicated the efflux pump protein might involve the transportation of GAS. The Papp of GAS was significant increased but the ER of GAS was significant decreased when co-administrated with Ver (P<0.05). The Papp of GAS was slightly increased and the ER of GAS was slightly decreased when co-administrated with Probenecid, while there was no significance. Conclusion: The results indicate that GAS is the substrate of P-gp. However, whether GAS is the substrate of MRP1 needs further research. Both Ver and Probenecid could enhance the transportation of GAS by competitive binding with P-gp and MRP1.

Influence of transcriptional factor Sp1 on expression of bile acids transporters MRP3 and MRP4 / 局解手术学杂志

Objective To investigate the influence of transcriptional factor Sp1 on expression of bile acids transporters MRP3 and MRP4 so as to perfect the regulatory mechanism of MRP3 and MRP4 expression.Methods Transformed Sp1-overexpression and Sp1 siRNA plas-mids to HepG2 cell and obtained the stably cells line.Then the expression levels of bile acids transporters MRP3 and MRP4 were measured by RT-qPCR,and the change of protein levels were detected by Western blot.Results The stably cells line Sp1-OE-HepG2 and Sp1siRNA-HepG2 were successfully transformed.The mRNA expression and protein levels of MRP3 and MRP4 were significantly increased in Sp1-OE-HepG2 cells,among which the mRNA expression of MRP3 mRNA increased 2.8 times,the protein levels of MRP3 increased 3.0 times,and the mRNA expression and protein levels of MRP4 increased 3.2 times and 2.5 times respectively.Conversely,the mRNA expression and protein levels of MRP3 and MRP4 were decreased in Sp1 siRNA-HepG2 cells,among which the mRNA expression of MRP3 mRNA de-creased 52%,the mRNA expression of MRP4 mRNA decreased 58%,the protein levels of MRP3 decreased 57%,and the protein levels of MRP4 decreased 60%.Conclusion Transcriptional factor Sp1 could regulate the expression of bile acids transporters MRP3 and MRP4 in HepG2 cells.

Comparative expression of MDR-1 and MRP in peripheral blood lymphocytes in lung cancer patients with chemotherapy treatment / 实用肿瘤学杂志

Objective To determine the significance of MDR -1 and MRP mRNA expression in periph-eral blood lymphocytes in lung cancer patients with chemotherapy treatment .Methods Peripheral blood samples were taken from each lung cancer patient before chemotherapy and three weeks after the first chemotherapy cycle . The expressions of MDR-1 and MRP were tested for 39 lung cancer cases using RT -PCR technique comparing to 20 normal control cases .Results There was no significant difference for MDR -1 mRNA and MRP mRNA ex-isting in lung cancer cases and control group (P>0.05).MDR-1 and MRP mRNA expressions were increased after treatment of chemotherapy courses in almost all pathological types of lung cancer .Furthermore,the expres-sion level in small cell lung cancer after chemotherapy was significantly higher than that before .Conclusion Chemotherapy may induce the incidence rate of MDR -1mRNA and MRP mRNA expression in lung cancer .Ade-nocarcinoma and squamous cell carcinoma are mainly intrinsic MDR while small cell lung cancer is mainly ac -quired MDR to chemotherapy .Based on the positive expression rate of MDR -1 mRNA and MRP mRNA in pe-ripheral blood ,we may evaluate the results of chemotherapy in lung cancer patients easily and conveniently .

Evaluation of reversal effect of 2-DG on multidrug resistance by detecting uptake of 99mTc-MIBI in HNE-1/DDP cells / 中国药理学通报

Aim To evaluate the reversal effect of 2-deoxy-D-glucose ( 2-DG ) on multidrug resistance ( MDR) by observing the uptake change of 99m Tc-MIBI in HNE-1/DDP cells, and to explore its mechanism. Methods The uptake of 99m Tc-MIBI in HNE-1/DDP cells under different concentrations of 2-DG was detec-ted by γ-counter, and the clearance rates of 99m Tc-MI-BI in HNE-1 cells and HNE-1/DDP cells after treated with 2-DG (10 mmol·L-1 ) were compared. The con-tent of ATP in HNE-1/DDP cells was detected after treated with 2-DG. P-glycoprotein ( P-gp ) and multi-drug resistance-associated proteins ( MRP ) expression were measured by Western blot. Apoptotic HNE-1/DDP cells treated with DDP alone or combined with 2-DG (10 mmol·L-1 ) were detected by propidium io-dide ( PI ) staining. Results The clearance rate of 99m Tc-MIBI in HNE-1/DDP cells was 54. 8%, which was significantly higher than that ( - 41. 3%) in HNE-1 cells (P<0. 01). The clearance rate of 99mTc-MIBI in HNE-1/DDP cells was -203. 7% after treat-ment with 2-DG ( 10 mmol · L-1 ) , which could be significantly reduced compared with the control group ( P<0. 01 ) . The level of ATP was 55 . 69% compared with the negative control group and the expression of P-gp and MRP protein decreased dramatically in HNE-1/DDP. With the combination of 2-DG and DDP, the ap-optotic rate of HNE-1/DDP cells reached 49 . 4%which was significantly higher than DDP treated group (22. 5%) . Conclusion Multidrug resistance and the reversal effect of 2-DG on multidrug resistance could be evaluated effectively by detecting the uptake change of 99m Tc-MIBI in HNE-1/DDP cells. The mechanism may be related with the inhibition of ATP level and the re-duced expression of P-gp and MRP protein in cancer cells.

Psoralen induced bile acid accumulation and cytotoxicity by inhibiting MRP2 and MRP3 in HepG2 cells / 中国药理学通报

Aim To investigate the toxicity of isopsor-alen in HepG2 cells and its effects on bile acid, bile acid synthesis and transport. Methods Cell viability was evaluated by MTT assay and bile acid was deter-mined inside HepG2 cells, with exposure to various isopsoralen for 24h. The mRNA transcription of BSEP, MRP2, MRP3, NTCP, OATP2, OSTα, CYP7A1, CYP27 A1 , FXR and PXR were assessed by real-time PCR. Results The cell viability was decreased dose-dependently with isopsoralen in HepG2 cells, and IC50 was 118. 1μmol·L-1 exposure to isopsoralen for 24h. Bile acid inside cells significantly increased with 100 and 400 μmol · L-1 isopsoralen. Isopsoralen caused the down-regulation of MRP2 , MRP3 , CYP7 A1 mRNA at 25 μmol · L-1 . Beside these, the up-regulation of OATP2,OSTα,CYP27A1,FXR,PXR with 100 μmol· L-1 isopsoralen, but there was no significant change of BSEP and NTCP. Conclusion The results show that isopsoralen induces bile acid accumulation and cytotox-icity which may be associated with the down-regulation of MRP2, MRP3 in HepG2 cells.

The expression of ABCC4/MRP4 and ABCC5/MRP5 gene in the NK/T cell lymphoma and its relationship with clinical efifcacy / 中国癌症杂志

Background and purpose: Natural killer/T cell lymphoma in poor effects, the production of multidrug resistance is one of the reasons to reduce the chemotherapy effect or failure. This study aimed to discuss the multidrug resistance associated protein 4 (ABCC4/MRP4) gene and multidrug resistance associated protein 5 (ABCC5/MRP5) gene expression in NK/T cell lymphoma SNK-6, YTS cell lines and NK/T cell lymphoma tissues, and the relationship between the level of ABCC4/MRP4, ABCC5/MRP5 gene expression and the clinical efifcacy. Methods:Real-time lfuorescence quantitative PCR (Real time-PCR) and immunohistochemical method (IHC) were used to detect the ABCC4/MRP4, ABCC5/MRP5 gene and protein expression. Results:Compared with the normal NK cells, ABCC4/MRP4 and ABCC5/MRP5 gene in SNK-6, YTS cell lines were highly expressed (P<0.05); Compared with rhinitis tissues, the expression of ABCC4/MRP4, ABCC5/MRP5 gene was higher in the NK/T cell lymphoma tissues (P<0.05);The expression level of ABCC4/MRP4 and ABCC5/MRP5 gene was negative correlation with clinical efifcacy (P<0.05). Conclusion: The expression of ABCC4/MRP4 and ABCC5/MRP5 gene affects the of clinical efifcacy of NK/T cell lymphoma.

MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.