Organ Specific Differences in Alteration of Aquaporin Expression in Rats Treated with Sennoside A, Senna Anthraquinones and Rhubarb Anthraquinones.

Senna and rhubarb are often used as routine laxatives, but there are differences in mechanism of action and potential side effects. Here, we studied metabolites of senna anthraquinones (SAQ), rhubarb anthraquinones (RAQ) and their chemical marker, sennoside A (SA), in a rat diarrhea model. In in vitro biotransformation experiments, SAQ, RAQ and SA were incubated with rat fecal flora solution and the metabolites produced were analyzed using HPLC. In in vivo studies, the same compounds were investigated for purgation induction, with measurement of histopathology and Aqps gene expression in six organs. The results indicated that SAQ and RAQ had similar principal constituents but could be degraded into different metabolites. A similar profile of Aqps down-regulation for all compounds was seen in the colon, suggesting a similar mechanism of action for purgation. However, in the kidneys and livers of the diarrhea-rats, down-regulation of Aqps was found in the RAQ-rats whereas up-regulation of Aqps was seen in the SAQ-rats. Furthermore, the RAQ-rats showed lower Aqp2 protein expression in the kidneys, whilst the SA-rats and SAQ-rats had higher Aqp2 protein expression in the kidneys. This may have implications for side effects of SAQ or RAQ in patients with chronic kidney or liver diseases.

Neuroprotective effect of Apelin 13 on ischemic stroke by activating AMPK/GSK-3ß/Nrf2 signaling.

BACKGROUND: Previous studies had showed that Apelin 13 could protect against apoptosis induced by ischemic/reperfusion (I/R). However, the mechanisms whereby Apelin 13 protected brain I/R remained to be elucidated. The present study was designed to determine whether Apelin 13 provided protection through AMPK/GSK-3ß/Nrf2 pathway. METHODS: In vivo, the I/R model was induced and Apelin 13 was given intracerebroventricularly 15 min before reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. For in vitro study, PC12 cells were used. To clarify the mechanisms, proteases inhibitors or siRNA were used. Protein levels were investigated by western blotting. RESULTS: The results showed that Apelin 13 treatment significantly reduced infarct size, improved neurological outcomes, decreased brain edema, and inhibited cell apoptosis, oxidative stress, and neuroinflammation after I/R. Apelin 13 significantly increased the expression of Nrf2 and the phosphorylation levels of AMPK and GSK-3ß. Furthermore, in cultured PC12 cells, the same protective effects were also observed. Silencing Nrf2 gene with its siRNA abolished the Apelin 13's prevention of I/R-induced PC12 cell injury, oxidative stress, and inflammation. Inhibition of AMPK by its siRNA decreased the level of Apelin 13-induced Nrf2 expression and diminished the protective effects of Apelin 13. The interplay relationship between GSK-3ß and Nrf2 was also verified with relative overexpression. Using selective inhibitors, we further identified the upstream of AMPK/GSK-3ß/Nrf2 is AR/Gα/PLC/IP3/CaMKK. CONCLUSIONS: In conclusion, the previous results showed that Apelin 13 protected against I/R-induced ROS-mediated inflammation and oxidative stress through activating the AMPK/GSK-3ß pathway by AR/Gα/PLC/IP3/CaMKK signaling, and further upregulated the expression of Nrf2-regulated antioxidant enzymes.

Protective effect of gentiopicroside from Gentiana macrophylla Pall. in ethanol-induced gastric mucosal injury in mice.

Gentiopicroside isolated from gentiana macrophylla Pall. belongs to iridoid glycosides. This study aimed to evaluate the protective effect of gentiopicroside against ethanol-induced gastric mucosal injury in mice. Mice were proactively administrated with gentiopicroside by intragastric administration once a day for 3 consecutive days. On the 3rd day, gastric ulcer in mice was induced with 70% ethanol after the last intragastric administration. The stomach tissues were submitted for evaluation of the severity of gastric mucosal alterations. Gentiopicroside administrated orally ameliorated the severity of gastric mucosal alterations. Oral administration of gentiopicroside significantly increased heat shock protein-70 and glutathione levels and superoxide dismutase activity, normalized epidermal growth factor and vascular endothelial growth factor levels, and decreased the levels of tumour necrosis factor-α, interleukin-6 and malondialdehyde, and myeloperoxidase activity in gastric tissue. These findings demonstrated that gentiopicroside has protective effect against ethanol-induced gastric mucosal injury in mice through the improvements of antioxidative and anti-inflammatory effects, as well as up-regulation of heat shock protein-70 level and normalization of epidermal growth factor and vascular endothelial growth factor levels. The results presented in this study provide some evidence for the development of a novel antigastric ulcer agent.

Aquaporins Alteration Profiles Revealed Different Actions of Senna, Sennosides, and Sennoside A in Diarrhea-Rats.

Senna and its main components sennosides are well-known effective laxative drugs and are used in the treatment of intestinal constipation in the world. Their potential side effects have attracted more attention in clinics but have little scientific justification. In this study, senna extract (SE), sennosides (SS), and sennoside A (SA) were prepared and used to generate diarrhea rats. The diarrhea rats were investigated with behaviors, clinical signs, organ index, pathological examination, and gene expression on multiple aquaporins (Aqps) including Aqp1, Aqp2, Aqp3, Aqp4, Aqp5, Aqp6, Aqp7, Aqp8, Aqp9, and Aqp11. Using qRT-PCR, the Aqp expression profiles were constructed for six organs including colon, kidney, liver, spleen, lung, and stomach. The Aqp alteration profiles were characterized and was performed with Principle Component Analysis (PCA). The SE treatments on the rats resulted in a significant body weight loss (p < 0.001), significant increases (p < 0.001) on the kidney index (27.72%) and liver index (42.55%), and distinguished changes with up-regulation on Aqps expressions in the kidneys and livers. The SS treatments showed prominent laxative actions and down regulation on Aqps expression in the colons. The study results indicated that the SE had more influence/toxicity on the kidneys and livers. The SS showed more powerful actions on the colons. We suggest that the caution should be particularly exercised in the patients with kidney and liver diseases when chronic using senna-based products.

The cornel Iridoid glycoside attenuated brain edema of the cerebral ischemia/reperfusion rats by modulating the polarized aquaporin 4.

Brain edema after ischemic stroke could worsen cerebral injury in patients who received intravenous thrombolysis. Cornus officinalis Sieb. et Zucc., a long-established traditional Chinese medicine, is beneficial to the treatment of neurodegenerative diseases including ischemic stroke. In particular, its major component, cornel iridoid glycoside (CIG), was evidenced to exhibit neuroprotective effects against cerebral ischemic/reperfusion injury (CIR/I). Aimed to explore the effects of the CIG on brain edema of the CIR/I rats, the CIG was analyzed with the main constituents by using HPLC. The molecular docking analysis was performed between the CIG constituents and AQP4-M23. TGN-020, an AQP4 inhibitor, was used as a comparison. In the in vivo experiments, the rats were pre-treated with the CIG and were injured by performing middle cerebral artery occlusion/reperfusion (MCAO/R). After 24 h, the rats were examined for neurological function, pathological changes, brain edema, and polarized Aqp4 expressions in the brain. The HPLC analysis indicated that the CIG was composed of morroniside and loganin. The molecular docking analysis showed that both morroniside and loganin displayed lower binding energies to AQP4-M23 than TGN-020. The CIG pre-treated rats exhibited fewer neurological function deficits, minimized brain swelling, and reduced lesion volumes compared to the MCAO/R rats. In the peri-infarct and infarct regions, the CIG pre-treatment restored the polarized Aqp4 expression which was lost in the MCAO/R rats. The results suggested that the CIG could attenuate brain edema of the cerebral ischemia/reperfusion rats by modulating the polarized Aqp4 through the interaction of AQP4-M23 with morroniside and loganin.

Aquaporins alteration revealed kidney damages in cerebral ischemia/reperfusion rats.

Background: Restoration of blood supply is a desired goal for the treatment of acute ischemic stroke. However, the restoration often leads to cerebral ischemia-reperfusion injury (CIR/I), which greatly increases the risk of non-neural organ damage. In particular, the acute kidney injury might be one of the most common complications. Aims: The study aimed to understand the damage occurred and the potential molecular mechanisms. Methods: The study was explored on the CIR/I rats generated by performing middle cerebral artery occlusion/reperfusion (MCAO/Reperfusion). The rats were evaluated with injury on the brains, followed by the non-neural organs including kidneys, livers, colons and stomachs. They were examined further with histopathological changes, and gene expression alterations by using RT-qPCR of ten aquaporins (Aqps) subtypes including Aqp1～Aqp9 and Aqp11. Furthermore, the Aqps expression profiles were constructed for each organ and analyzed by performing Principle Component Analysis. In addition, immunohistochemistry was explored to look at the protein expression of Aqp1, Aqp2, Aqp3 and Aqp4 in the rat kidneys. Results: There was a prominent down-regulation profile in the MCAO/Reperfusion rat kidneys. The protein expression of Aqp1, Aqp2, Aqp3 and Aqp4 was decreased in the kidneys of the MCAO/Reperfusion rats. We suggested that the kidney was in the highest risk to be damaged following the CIR/I. Down-regulation of Aqp2, Aqp3 and Aqp4 was involved in the acute kidney injury induced by the CIR/I.

Sennoside A inhibits quorum sensing system to attenuate its regulated virulence and pathogenicity via targeting LasR in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen, and the emergence of drug resistance greatly increased the difficulty of treating its infection. Cell density-dependent quorum sensing (QS) system not only regulates the virulence but also associates with the drug resistance of P. aeruginosa. Screening for agents targeting QS to inhibit bacterial virulence and pathogenicity is considered a promising strategy to combat P. aeruginosa infection. In the present study, sennoside A was found to be able to inhibit the QS expression of P. aeruginosa at subinhibitory concentrations. The QS-regulated virulence factors, including protease, elastase, rhamnolipid, and pyocyanin, were also inhibited by sennoside A at both transcriptional and translational levels. Moreover, sennoside A could suppress the motility of twitching, swimming, and swarming as well as the biofilm formation, which is associated with the acute and chronic infections of P. aeruginosa in a dose-dependent manner. The attenuated pathogenicity of P. aeruginosa by sennoside A was further verified by Chinese cabbage, Drosophila melanogaster, and Caenorhabditis elegans infection analysis. Further study found that sennoside A might target the las system, mainly LasR, to interfere with QS. All the results indicate that sennoside A could inhibit the QS system to attenuate its regulated virulence and pathogenicity via mainly targeting LasR in P. aeruginosa and further research to identify its anti-QS activity for other Gram-negative bacteria is warranted.

In vitro functional effects of XPC gene rare variants from bladder cancer patients.

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients' germ line DNA. In a case-control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3' untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0-9.8, P=0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3' UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts.

Functional assays to determine the significance of two common XPC 3'UTR variants found in bladder cancer patients.

BACKGROUND: XPC is involved in the nucleotide excision repair of DNA damaged by carcinogens known to cause bladder cancer. Individuals homozygous for the variant allele of XPC c.1496C > T (p.Ala499Val) were shown in a large pooled analysis to have an increased bladder cancer risk, and we found two 3'UTR variants, *611T > A and c.*618A > G, to be in strong linkage disequilibrium with c.1496T. Here we determined if these two 3'UTR variants can affect mRNA stability and assessed the impact of all three variants on mRNA and protein expression. METHODS: In vitro mRNA stability assays were performed and mRNA and protein expression measured both in plasmid-based assays and in lymphocytes and lymphoblastoid cell lines from bladder and breast cancer patients. RESULTS: The two 3'UTR variants were associated with reduced protein and mRNA expression in plasmid-based assays, suggesting an effect on mRNA stability and/or transcription/translation. A near-significant reduction in XPC protein expression (p = 0.058) was detected in lymphoblastoid cell lines homozygous for these alleles but no differences in mRNA stability in these lines was found or in mRNA or protein levels in lymphocytes heterozygous for these alleles. CONCLUSION: The two 3'UTR variants may be the variants underlying the association of c.1496C > T and bladder cancer risk acting via a mechanism modulating protein expression.

Identification of rhein as the metabolite responsible for toxicity of rhubarb anthraquinones.

Rhubarb is a popular food in Europe with laxative properties attributed to anthraquinones. Long term usage of rhubarb anthraquinones has been linked to colonic toxicity, including the formation of melanosis coli, which is associated with increased risk of colon cancer. The major purgative anthraquinone in rhubarb is thought to be sennoside A, which is metabolised by colonic microflora. Here, we sought to identify the toxic metabolite responsible for melanosis coli in rats dosed with rhubarb anthraquinones for up to 90 days. Three metabolites were detected in rat faeces using HPLC. Of these, rhein was identified as the metabolite that accumulated most over time. Fecal flora from treated rats were capable of greater biotransformation of sennoside A to rhein compared to that from control rats. Cell culture experiments suggested that apoptosis and autophagy induced by rhein is the likely mechanism of chronic toxicity of rhubarb anthraquinones.

Chikusetsu saponin IVa protects pancreatic ß cell against intermittent high glucose-induced injury by activating Wnt/ß-catenin/TCF7L2 pathway.

Islet ß cell mass reduction induced by glucose fluctuation is crucial for the development and progression of T2DM. Chikusetsu saponin IVa (CHS) had protective effects against DM and related injuries. Here we aimed to investigate the role of CHS in ß cell injuries and its possible mechanism involved. Isolated rat islets, ßTC3 cells and T2DM mice were used in this study. The results showed that CHS restored the secretion activity, promoted ß cell survival by increasing ß cell proliferation and decreasing apoptosis which induced by intermittent high glucose (IHG). In vivo, CHS protected ß cell apoptosis to normalize blood glucose and improve insulin sensitivity in DM mice. Further studies showed that CHS activated Wnt3a signaling, inhibited HBP1, promoted ß-catenin nuclear translocation, enhanced expressions of TCF7L2, GIPR and GLP-1R, inhibited p53, p27 and p21. The protective effect of CHS was remarkably suppressed by siRNAs against TCF7L2 or XAV-939 (a Wnt/ß-catenin antagonist) in vitro and in ß-catenin-/- mice. In conclusion, we identified a novel role of CHS in protecting ß cell survival and regeneration by mechanisms involving the activation of Wnt3a/ß-catenin/TCF7L2 signaling. Our results indicated the potential value of CHS as a possible intervention drug for T2DM.

Amelioration of nonalcoholic fatty liver disease by swertiamarin in fructose-fed mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease. Swertia bimaculata (Sieb. et Zucc.) Hook. Thoms.ex Clarke, a glabrous or procumbent perennial herb, is a traditional herb medicine. Swertiamarin, a secoiridoid glycoside, is a representative ingredient in this medical plant crude extract and shows antidiabetic and antihyperlipidaemic activities and protective effect against hepatic injury. PURPOSE: The present study aimed to determine whether swertiamarin can attenuate NAFLD in fructose-fed mice. METHODS: Healthy male mice freely drank water containing 10% fructose for 12 consecutive weeks, whereas animals in those swertiamarin tested groups received different doses of swertiamarin (25, 50 and 100 mg/kg) by intragastric administration once a day from the ninth week to the twelfth week. RESULTS: At the end of the experiment, fructose-fed mice administrated with swertiamarin showed low levels of serum glucose, triglycerides, uric acid, alanine aminotransferase and aspartate transaminase. Histological examinations suggested the alleviation of hepatic ballooning degeneration and steatosis by swertiamarin treatment. Moreover, swertiamarin administration mitigated hepatic oxidative stress along with decreases of hepatic pro-inflammation cytokines, which was associated with decrease of hepatic xanthine oxidase (XO) activity and enhancements of anti-oxidant defense system enzymes, as well as activation of nuclear factor E2-related factor 2 (Nrf2) in fructose-fed mice. In addition, swertiamarin down-regulated expression of sterol-regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC1) in liver of fructose-fed mice. CONCLUSION: The present study demonstrates that swertiamarin alleviates NAFLD and metabolic alterations in fructose-fed mice.

Aralia taibaiensis Protects against I/R-Induced Brain Cell Injury through the Akt/SIRT1/FOXO3a Pathway.

BACKGROUND: Saponin from Aralia taibaiensis (sAT) showed excellent antioxidative effects in several models; however, its effects on brain cells were unknown to us. The present study was designed to evaluate the protective effects of sAT on ischemia/reperfusion- (I/R-) induced injury and clarify its mechanisms. METHODS: In vitro, HT22 cells were pretreated with sAT and then subjected to I/R. Apoptosis rate, mitochondrial function, and antioxidant proteins were measured. To clarify the mechanisms, siRNA were used. In vivo, sAT was pretreated through intragastric administration for 7 days and the I/R model was induced. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. Protein levels were investigated by Western blotting. RESULTS: The results showed that sAT treatment significantly protected cells from I/R-induced cell apoptosis and mitochondrial dysfunction. The antioxidant protein levels were increased in a dose-dependent manner. Further study revealed that sAT induced the deacetylation and phosphorylation of PGC-1α and FOXO3a. sAT treatment also induced the phosphorylation levels of Akt and the expression levels of SIRT1. Using the specific targeted siRNA transfection, the interplay relationship between Akt, SIRT1, PGC-1α, and FOXO3a was verified. Furthermore, the same protective effects were also observed in rats subjected to I/R. CONCLUSION: sAT protected brain cells from I/R-induced mitochondrial oxidative stress and dysfunction through regulating the Akt/SIRT1/FOXO3a/PGC-1α pathway.

Imatinib radiosensitizes bladder cancer by targeting homologous recombination.

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. ©2012 AACR.