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Inhibition of P-glycoprotein Gene Expression and Function Enhances Triptolide-induced Hepatotoxicity in Mice.

Kong LL, Zhuang XM, Yang HY, Yuan M, Xu L, Li H - Sci Rep (2015)

Bottom Line: Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes.The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased.P-gp mediated clearance played a significant role in TP detoxification.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Toxicology and Medical Countermeasures, Beijing 100850, China [2] Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.

ABSTRACT
Triptolide (TP) is the major active principle of Tripterygium wilfordii Hook f. and very effective in treatment of autoimmune diseases. However, TP induced hepatotoxicity limited its clinical applications. Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes. In this study, small interfering RNA (siRNA) and specific inhibitor tariquidar were used to investigate the impact of P-gp down regulation on TP-induced hepatotoxicity. The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers (ALT and AST) and pathological changes in liver. The other toxicological indicators (MDA, SOD and Bcl-2/Bax) were also significantly changed by P-gp inhibition. The data analysis showed that the increase of TP exposure in mice was quantitatively correlated to the enhanced hepatotoxicity, and the hepatic exposure was more relevant to the toxicity. P-gp mediated clearance played a significant role in TP detoxification. The risk of herb-drug interaction likely occurs when TP is concomitant with P-gp inhibitors or substrates in clinic.

No MeSH data available.


Related in: MedlinePlus

Screening and functional evaluation of mdr1a siRNA in vitro and in vivo.(a) RNAi-mediated knockdown of mdr1a mRNA in CT26.WT cells. (b) RNAi-mediated knockdown of P-gp protein in CT26.WT cells. CT26.WT cells were transfected with mdr1a siRNAs. After 48 h, mRNA for mdr1a gene and P-gp protein expression were measured by real-time PCR and western blot. (c) RNAi-mediated knockdown of P-gp protein expression in liver of mice. mdr1a-1 siRNA (at a dose of 5, 10, 15 nmol) was injected via the tail vein of mice. After 48 h, the livers of mice were collected and the P-gp protein expression was measured to validate the efficacy of siRNA. (d) Evaluation of P-gp function after pretreatment with mdr1a-1 siRNA in mice. The NC-siRNA or mdr1a-1 siRNA (15 nM) was intravenously injected 2 days prior to the digoxin injection at a dose of 1 mg/kg. The blood samples were collected at scheduled time intervals. Data are presented as mean ± SD (n = 3 in vitro and n = 5 in vivo). #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC-siRNA group.
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f2: Screening and functional evaluation of mdr1a siRNA in vitro and in vivo.(a) RNAi-mediated knockdown of mdr1a mRNA in CT26.WT cells. (b) RNAi-mediated knockdown of P-gp protein in CT26.WT cells. CT26.WT cells were transfected with mdr1a siRNAs. After 48 h, mRNA for mdr1a gene and P-gp protein expression were measured by real-time PCR and western blot. (c) RNAi-mediated knockdown of P-gp protein expression in liver of mice. mdr1a-1 siRNA (at a dose of 5, 10, 15 nmol) was injected via the tail vein of mice. After 48 h, the livers of mice were collected and the P-gp protein expression was measured to validate the efficacy of siRNA. (d) Evaluation of P-gp function after pretreatment with mdr1a-1 siRNA in mice. The NC-siRNA or mdr1a-1 siRNA (15 nM) was intravenously injected 2 days prior to the digoxin injection at a dose of 1 mg/kg. The blood samples were collected at scheduled time intervals. Data are presented as mean ± SD (n = 3 in vitro and n = 5 in vivo). #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC-siRNA group.

Mentions: In this study, CT26.WT cells were transfected with various siRNA sequences against mdr1a. The suppressive effect was measured at 48 h after transfection to evaluate the specificity of siRNA targeted to mdr1a. Figure 2a showed the silencing efficiency of three siRNA sequences on the same mRNA target. All the three siRNA sequences inhibited the expression of mdr1a mRNA. However significant differences in silencing efficiency were observed, with 63%, 51% and 46% for mdr1a-1, mdr1a-2 and mdr1a-3 siRNA, respectively. Western blot assay further indicated that the P-gp expression in the cells treated with these mdr1a siRNA was also remarkably reduced (Fig. 2b). Among the three sequences, mdr1a-1 was the most efficient one for reducing both the expression of mdr1a mRNA and protein. In addition, the protein expressions of BCRP and MRP2 were not changed, which confirmed the specificity of mdr1a-1 siRNA (Fig. 2b). Therefore, mdr1a-1 siRNA was selected for the in vivo study.


Inhibition of P-glycoprotein Gene Expression and Function Enhances Triptolide-induced Hepatotoxicity in Mice.

Kong LL, Zhuang XM, Yang HY, Yuan M, Xu L, Li H - Sci Rep (2015)

Screening and functional evaluation of mdr1a siRNA in vitro and in vivo.(a) RNAi-mediated knockdown of mdr1a mRNA in CT26.WT cells. (b) RNAi-mediated knockdown of P-gp protein in CT26.WT cells. CT26.WT cells were transfected with mdr1a siRNAs. After 48 h, mRNA for mdr1a gene and P-gp protein expression were measured by real-time PCR and western blot. (c) RNAi-mediated knockdown of P-gp protein expression in liver of mice. mdr1a-1 siRNA (at a dose of 5, 10, 15 nmol) was injected via the tail vein of mice. After 48 h, the livers of mice were collected and the P-gp protein expression was measured to validate the efficacy of siRNA. (d) Evaluation of P-gp function after pretreatment with mdr1a-1 siRNA in mice. The NC-siRNA or mdr1a-1 siRNA (15 nM) was intravenously injected 2 days prior to the digoxin injection at a dose of 1 mg/kg. The blood samples were collected at scheduled time intervals. Data are presented as mean ± SD (n = 3 in vitro and n = 5 in vivo). #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC-siRNA group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Screening and functional evaluation of mdr1a siRNA in vitro and in vivo.(a) RNAi-mediated knockdown of mdr1a mRNA in CT26.WT cells. (b) RNAi-mediated knockdown of P-gp protein in CT26.WT cells. CT26.WT cells were transfected with mdr1a siRNAs. After 48 h, mRNA for mdr1a gene and P-gp protein expression were measured by real-time PCR and western blot. (c) RNAi-mediated knockdown of P-gp protein expression in liver of mice. mdr1a-1 siRNA (at a dose of 5, 10, 15 nmol) was injected via the tail vein of mice. After 48 h, the livers of mice were collected and the P-gp protein expression was measured to validate the efficacy of siRNA. (d) Evaluation of P-gp function after pretreatment with mdr1a-1 siRNA in mice. The NC-siRNA or mdr1a-1 siRNA (15 nM) was intravenously injected 2 days prior to the digoxin injection at a dose of 1 mg/kg. The blood samples were collected at scheduled time intervals. Data are presented as mean ± SD (n = 3 in vitro and n = 5 in vivo). #P < 0.05, ##P < 0.01, ###P < 0.001 compared with NC-siRNA group.
Mentions: In this study, CT26.WT cells were transfected with various siRNA sequences against mdr1a. The suppressive effect was measured at 48 h after transfection to evaluate the specificity of siRNA targeted to mdr1a. Figure 2a showed the silencing efficiency of three siRNA sequences on the same mRNA target. All the three siRNA sequences inhibited the expression of mdr1a mRNA. However significant differences in silencing efficiency were observed, with 63%, 51% and 46% for mdr1a-1, mdr1a-2 and mdr1a-3 siRNA, respectively. Western blot assay further indicated that the P-gp expression in the cells treated with these mdr1a siRNA was also remarkably reduced (Fig. 2b). Among the three sequences, mdr1a-1 was the most efficient one for reducing both the expression of mdr1a mRNA and protein. In addition, the protein expressions of BCRP and MRP2 were not changed, which confirmed the specificity of mdr1a-1 siRNA (Fig. 2b). Therefore, mdr1a-1 siRNA was selected for the in vivo study.

Bottom Line: Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes.The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased.P-gp mediated clearance played a significant role in TP detoxification.

View Article: PubMed Central - PubMed

Affiliation: 1] State Key Laboratory of Toxicology and Medical Countermeasures, Beijing 100850, China [2] Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.

ABSTRACT
Triptolide (TP) is the major active principle of Tripterygium wilfordii Hook f. and very effective in treatment of autoimmune diseases. However, TP induced hepatotoxicity limited its clinical applications. Our previous study found that TP was a substrate of P-glycoprotein and its hepatobiliary clearance was markedly affected by P-gp modulation in sandwich-cultured rat hepatocytes. In this study, small interfering RNA (siRNA) and specific inhibitor tariquidar were used to investigate the impact of P-gp down regulation on TP-induced hepatotoxicity. The results showed that when the function of P-gp was inhibited by mdr1a-1 siRNA or tariquidar, the systemic and hepatic exposures of TP were significantly increased. The aggravated hepatotoxicity was evidenced with the remarkably lifted levels of serum biomarkers (ALT and AST) and pathological changes in liver. The other toxicological indicators (MDA, SOD and Bcl-2/Bax) were also significantly changed by P-gp inhibition. The data analysis showed that the increase of TP exposure in mice was quantitatively correlated to the enhanced hepatotoxicity, and the hepatic exposure was more relevant to the toxicity. P-gp mediated clearance played a significant role in TP detoxification. The risk of herb-drug interaction likely occurs when TP is concomitant with P-gp inhibitors or substrates in clinic.

No MeSH data available.


Related in: MedlinePlus