Xianglin Tang

The protective effect of piperine on dextran sulfate sodium induced inflammatory bowel disease and its relation with pregnane X receptor activation

ETHNOPHARMACOLOGICAL RELEVANCE Inflammatory bowel disease (IBD) is associated with chronic inflammation of the intestinal tract. Piperine (1-peperoylpiperidine), the primary lipophilic component in black pepper (Piper nigrum) and long pepper (Piper longum), has been reported to be effective for anti-inflammatory. Rencently, several ethnopharmacological purity compounds, such as baicalin and artemisinin, are reported to have potentially therapeutic role in treating IBD. In the present study, the effects of piperine on pregnane X receptor (PXR)-mediated CYP3A expression and its therapeutic role in IBD were investigated. MATERIALS AND METHODS LS174T cells and C57BL/6J mice were treated by the piperine. Gene expressions were analyzed by real-time PCR, Western blot analysis, transient transfections assay and histological analysis. RESULTS Data indicated that treatment of LS174T cells with piperine markedly increased both CYP3A4 and PXR mRNA and protein. Transient transfection experiments indicated that transcriptional activation of the CYP3A4 gene via piperine was PXR-dependent. Data show that pre-administration of piperine decreased clinical hallmarks of colitis in DSS-treated PXR mice as measured by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. Inflammatory mediators (CCR2, ICAM-1, IL-1β, IL-6, IL-10, iNOS, MCP-1, and TNFα) after DSS treatment were significantly decreased in mice pretreated with piperine but corresponding conditions did not occur in mice with down-regulation of PXR by small interfering RNA (siRNA). CONCLUSION Piperine is a potential agonist of PXR and an inducer of PXR, which may induce CYP3A4 gene expression at the mRNA and protein levels. These results establish that piperine may contribute to prevention or reduction of colonic inflammation.

Tanshinone IIA exerts protective effects in a LCA-induced cholestatic liver model associated with participation of pregnane X receptor.

Tanshinone IIA (Tan IIA) is one of the main natural active ingredients purified from Salvia miltiorrhiza radix, which has long been used in clinical practice in China to treat diseases including liver fibrosis, Alzheimer׳s disease, and cardiovascular diseases. Tan IIA has hepatoprotective properties, and is an efficacious PXR agonist. Our study was designed to observe the function and mechanism of the hepatoprotective properties of Tan IIA. HepG2 cells were used to investigate the vitrol effects of Tan IIA on PXR and CYP3A4. Gut-formed LCA is hepatotoxic, and has been implicated in the pathogenesis of cholestatic diseases. To further investigate the hepatoprotective mechanisms of Tan IIA against LCA-induced cholestasis in vivo, we choose the normal mice and siRNA-treated mice. The in vitro study demonstrated that the effect of Tan IIA on CYP3A4 was mediated by transactivation of PXR in a dose- and time-dependent manner. The in vivo experiments using PXR siRNA revealed that Tan IIA could protect against LCA-induced hepatotoxicity and cholestasis in a dose-dependent manner. These effects were partially caused by the upregulation of PXR, as well as Cyp3a11, Cyp3a13, and Mdr1, which are the enzymes responsible for LCA metabolism. This is the first report showing that the hepatoprotective effects of Tan IIA are partly mediated by PXR.

Artemisinin protects against dextran sulfate-sodium-induced inflammatory bowel disease, which is associated with activation of the pregnane X receptor

Artemisinin has been used to treat malaria for centuries in the context of traditional Chinese medicine. In the present study, the effects of artemisinin on pregnane X receptor (PXR)-mediated CYP3A expression and its therapeutic role in inflammatory bowel disease were investigated. LS174T cells exposed to artemisinin at various concentrations and for different periods of time were examined with respect to the specific induction of CYP3A4 and PXR mRNA expression. Transient transfection experiments showed transcriptional activation of the CYP3A4 gene through artemisinin to be PXR-dependent. An electrophoretic-mobility shift assay (EMSA) showed that artemisinin activates the DNA-binding capacity of the PXR for the CYP3A4 element. These results indicate that the induction of CYP3A4 by artemisinin is mediated through the activation of PXR. Using animal models, it was demonstrated that artemisinin abrogates dextran sulfate sodium (DDS)-induced intestinal inflammation. Preadministration of artemisinin ameliorated the clinical hallmarks of colitis in DSS-treated mice as determined by body weight loss and assessment of diarrhea, rectal bleeding, colon length, and histology. Artemisinin was found to prevent or reduce the severity of colonic inflammation by inducing CYP3A expression by activation of PXR.

In vivo metabolite identification of bakuchiol in rats by UPLC/ESI-PDA-QTOF-MS.

Psoralea corylifolia L. is a traditional Chinese medicine for the treatment of various skin diseases such as psoriasis, vitiligo and chronic graft-versus-host. Bakuchiol, isolated from the seeds of P. corylifolia L., is one of the most important active compounds. To study the metabolic fate of bakuchiol in rats, an ultra performance liquid chromatography/electrospray ionization-photo diode array-quadrupole time of flight-mass spectrometry (UPLC/ESI-PDA-QTOF-MS) combined with MetaboLynx XS software was used, peak area-time curves of major metabolites in plasma from bakuchiol were determined. A total of 11 metabolites were identified after a single oral administration of bakuchiol, including 6 in plasma, 10 in bile, 8 in urine and 2 in feces, the metabolic transformation pathways of bakuchiol in rats included oxidation, hydroxylation, methylation, O-glucuronide conjugation and O-sulfate conjugation. In conclusion, this study expands our knowledge about the metabolism of bakuchiol which will be conducive to reveal its in vivo pharmacological and toxicological material basis, in addition, UPLC/ESI-PDA-QTOF-MS coupled with MetaboLynx XS software can be adopted as a useful tool for quick detection and identification of metabolites from medicine in vivo.

Ophiopogonin D maintains Ca2＋ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/ EETs and suppressing ER stress

Aim:CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis.Methods:Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca2+ concentrations ([Ca2+]i) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA.Results:Angiotensin II (10−6 mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca2+]i in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca2+ homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca2+ homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca2+ homeostasis. Furthermore, the intracellular Ca2+ chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells.Conclusion:OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca2+ homeostasis and ER stress.

Tetramethylpyrazine protects lymphocytes from radiation-induced apoptosis through nuclear factor-κB.

AIM Radiation induces an important apoptosis response in irradiated organs. The objective of this study was to investigate the radioprotective effect of tetramethylpyrazine (TMP) on irradiated lymphocytes and discover the possible mechanism of protection. METHOD Lymphocytes were pretreated for 12 h with TMP (25-200 μmol·L(-1)) and then exposed to 4 Gy radiation. Cell apoptosis and the signaling pathway were analyzed. RESULTS Irradiation increased cell death, DNA fragmentation, activated caspase activation and cytochrome c translocation, downregulated B-cell lymphoma 2 (Bcl-2) and up-regulated Bcl-2-associated X protein (Bax). Pretreated with TMP significantly reversed this tendency. Several anti-apoptotic characteristics of TMP, including the ability to increase cell viability, inhibit caspase-9 activation, and upregulate Bcl-2 and down-regulate Bax in 4Gy-irradiated lymphocytes were determined. Signal pathway analysis showed TMP could translate nuclear factor-κB (NF-κB) from cytosol into the nucleus. CONCLUSION The results suggest that TMP had a radioprotective effect through the NF-κB pathway to inhibit apoptosis, and it may be an effective candidate for treating radiation diseases associated with cell apoptosis.

Ginkgolide B protects human umbilical vein endothelial cells against xenobiotic injuries via PXR activation

Aim:Pregnane X receptor (PXR) is a nuclear receptor that regulates a number of genes encoding drug metabolism enzymes and transporters and plays a key role in xeno- and endobiotic detoxification. Ginkgolide B has shown to increase the activity of PXR. Here we examined whether ginkgolide B activated PXR and attenuated xenobiotic-induced injuries in endothelial cells.Methods:Human umbilical vein endothelial cells (HUVECs) were treated with ginkgolide B. The expression of PXR, CYP3A4, MDR1, VCAM-1, E-selectin and caspase-3 were quantified with qRT-PCR and Western blot analysis. Cell apoptosis was analyzed with flow cytometry. Fluorescently labeled human acute monocytic leukemia cells (THP-1 cells) were used to examine cell adhesion.Results:Ginkgolide B (30–300 μmol/L) did not change the mRNA and protein levels of PXR in the cells, but dose-dependently increased nuclear translocation of PXR protein. Ginkgolide B increased the expression of CYP3A4 and MDR1 in the cells, which was partially reversed by pretreatment with the selective PXR signaling antagonist sulforaphane, or transfection with PXR siRNA. Functionally, ginkgolide B dose-dependently attenuated doxorubicin- or staurosporine-induced apoptosis, which was reversed by transfection with PXR siRNA. Moreover, ginkgolide B suppressed TNF-α-induced THP-1 cell adhesion and TNF-α-induced expression of vascular adhesion molecule 1 (VCAM-1) and E-selectin in the cells, which was also reversed by transfection with PXR siRNA.Conclusion:Ginkgolide B exerts anti-apoptotic and anti-inflammatory effects on endothelial cells via PXR activation, suggesting that a PXR-mediated endothelial detoxification program may be important for protecting endothelial cells from xeno- and endobiotic-induced injuries.

Serum Pharmacochemistry Analysis Using UPLC-Q-TOF/MS after Oral Administration to Rats of Shenfu Decoction

The purpose of this study was to study the serum pharmacochemistry of SFD as well as the material basis through analyzing the constituents absorbed in blood. The SFD was orally administrated to Wistar rats at 20 g·kg−1, and Ultra Performance Liquid Chromatography (UPLC) fingerprints of SFD were created. Serum samples were collected for analysis, and further data processing used MarkerLynx XS software. 19 ginsenosides and 16 alkaloids were detected in SFD. The absorption of alkaloids (mainly monoester diterpenoid alkaloids) increased when Aconitum carmichaeli Debx. was combined with Panax ginseng, while the ginsenosides remained stable. Diester diterpenoid alkaloids were not present in the serum samples. A suitable serum pharmacochemistry method was successfully established to study pharmacological effects and potential improvements in formulation. This may also be useful for toxicity reduction. We suspect that the increased absorption of the monoester diterpenoid alkaloids from the mixture of Panax and Radix, compared to the Panax only extract, may be the reason for the combination of the two herbs in popular medicine formulas in China.

Tanshinone IIA ameliorates dextran sulfate sodium-induced inflammatory bowel disease via the pregnane X receptor

Tanshinone IIA (Tan IIA) (C19H18O3) is one of the major active lipophilic components in a conventional Chinese medicine called danshen, and it has long been used in the People’s Republic of China and other neighboring countries to treat patients suffering from inflammatory bowel disease (IBD). Previous experiments by many teams determined which mechanism of Tan IIA is relevant to the treatment of IBD associated with inflammation and the pregnane X receptor (PXR). The current study demonstrated that Tan IIA is an efficacious PXR agonist and its ability to induce CYP3A4 mRNA and protein expression was mediated by the transactivation of PXR, a known target of abrogating inflammation in IBD. Clinical symptoms in mice and histological assessment data suggested that administration of Tan IIA in mice demonstrated significant protection and showed that in DSS-induced IBD it acts in a concentration-dependent manner. PXR-silenced mice treated with Tan IIA demonstrated low protection against DSS-induced mouse IBD and exacerbated the severity of IBD compared with wild-type mice; PXR-silenced mice demonstrated the necessity for PXR in Tan IIA-mediated upregulation of xenobiotic metabolism genes. The IBD treatment effects of Tan IIA are partially due to PXR-mediated upregulation of xenobiotic metabolism and downregulation of inflammatory mediators. The novel findings reported here may contribute to the effective utilization of Tan IIA and its derivatives as a PXR ligand in the treatment of human IBD. This suggests that Tan IIA may have considerable clinical utility.

Rapid comparison of metabolites in humans and rats of different sexes using untargeted UPLC-TOFMS and an in-house software platform

Metabolite differences between sexes have rarely been observed in a global manner, but it has recently been made possible by the advancement in metabolomics techniques. In this study, untargeted ultraperformance liquid chromatography coupled to time-of-flight mass spectrometry and an in-house software platform were used for a rapid comparison of sex differences in urinary metabolites in humans and in urinary and serum metabolites in Sprague Dawley (SD) rats. In addition, the species differences of urinary metabolites between humans and SD rats were also observed. Principle component analysis showed that all the observed metabolite sex differences were more distinct in SD rats than in humans, indicating that the sex differences of human urinary metabolites is small compared with that of SD rats. In SD rats, the observed metabolite sex differences were more distinct in urine than in serum, indicating the importance of urine analysis for metabolomics studies. The species differences in the urinary metabolites of humans and SD rats were much more distinct than any of the observed sex differences. Many sex- and species-related markers were discovered and putatively identified. In both humans and SD rats, steroid metabolites appeared to constitute a major sex difference in urinary metabolites. This provides new proof of the special importance of steroid metabolites in sex differences from an untargeted metabolomics investigation, which is rare for sex differences. Contrary patterns involving adrenocortical activity appeared to exist between rodents and humans, which agrees with previous reports. In the serum metabolites of SD rats, sex differences in ascorbic acid or its isomer and pantothenic acid or its isomer, but not in steroid metabolites, were prominent. Human-specific α-N- phenylacetyl-l-glutamine and androsterone glucuronide were among the putative identities of the markers discriminating humans and SD rats. This study demonstrated the feasibility of an in-house software platform and provides metabolite-related information on sex and species differences.Metabolite differences between sexes have rarely been observed in a global manner, but it has recently been made possible by the advancement in metabolomics techniques. In this study, untargeted ultraperformance liquid chromatography coupled to time-of-flight mass spectrometry and an in-house software platform were used for a rapid comparison of sex differences in urinary metabolites in humans and in urinary and serum metabolites in Sprague Dawley (SD) rats. In addition, the species differences of urinary metabolites between humans and SD rats were also observed. Principle component analysis showed that all the observed metabolite sex differences were more distinct in SD rats than in humans, indicating that the sex differences of human urinary metabolites is small compared with that of SD rats. In SD rats, the observed metabolite sex differences were more distinct in urine than in serum, indicating the importance of urine analysis for metabolomics studies. The species differences in the urinary metabolites of humans and SD rats were much more distinct than any of the observed sex differences. Many sex- and species-related markers were discovered and putatively identified. In both humans and SD rats, steroid metabolites appeared to constitute a major sex difference in urinary metabolites. This provides new proof of the special importance of steroid metabolites in sex differences from an untargeted metabolomics investigation, which is rare for sex differences. Contrary patterns involving adrenocortical activity appeared to exist between rodents and humans, which agrees with previous reports. In the serum metabolites of SD rats, sex differences in ascorbic acid or its isomer and pantothenic acid or its isomer, but not in steroid metabolites, were prominent. Human-specific α-N-phenylacetyl-L-glutamine and androsterone glucuronide were among the putative identities of the markers discriminating humans and SD rats. This study demonstrated the feasibility of an in-house software platform and provides metabolite-related information on sex and species differences.