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Aim To investigate the hepatotoxicity of rutecarpine(RUT)by using high-content screening technology.Methods HepG2 cells were exposed to different concentrations of RUT for different time, then cell viability was detected by MTT method.Cell count, nucleus injury, mitochondrial membrane potential(MMP), reactive oxygen species(ROS), internal flow of calcium, cell membrane integrity(DIR)were measured by high-content screening technology.The activation of MAPK, NF-κB and JAKs-STATs was assayed by high-content screening technology.The apoptosis was detected by flow cytometry.Results The viability was significantly reduced by 100 μmol·L-1 RUT(P<0.01)after HepG2 cell exposure to RUT for 24 h, the nuclear area decreased and the nuclear morphology was uneven, and after 48 h, the cell count was significantly reduced(P<0.01), the early apoptosis was detected(P<0.01).After HepG2 cell exposure to RUT for 6 h, the levels of ROS and internal flow of calcium significantly increased(P<0.01), and the cell membrane integrity was obviously damaged(P<0.01).After exposure to 100 μmol·L-1 RUT for 24 h, the phosphorylation of ERK, JNK, STAT3 and p38 significantly increased(P<0.01, P<0.05), but there was no significant change in total protein level.The expression of c-Jun and c-Fos was up-regulated at 3 h(P<0.01), and at 3h time point, the phosphorylation of NF-κB p65 significantly increased(P<0.01), but nuclear translocation was not significant.Conclusions Under certain conditions, RUT shows cytotoxicity on HepG2 cells, and its toxic mechanism is mainly related to injury caused by oxidative stress and inflammatory response.
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Aim To investigate the hepatotoxicity of rutecarpine(RUT)by using high-content screening technology.Methods HepG2 cells were exposed to different concentrations of RUT for different time, then cell viability was detected by MTT method.Cell count, nucleus injury, mitochondrial membrane potential(MMP), reactive oxygen species(ROS), internal flow of calcium, cell membrane integrity(DIR)were measured by high-content screening technology.The activation of MAPK, NF-κB and JAKs-STATs was assayed by high-content screening technology.The apoptosis was detected by flow cytometry.Results The viability was significantly reduced by 100 μmol·L-1 RUT(P<0.01)after HepG2 cell exposure to RUT for 24 h, the nuclear area decreased and the nuclear morphology was uneven, and after 48 h, the cell count was significantly reduced(P<0.01), the early apoptosis was detected(P<0.01).After HepG2 cell exposure to RUT for 6 h, the levels of ROS and internal flow of calcium significantly increased(P<0.01), and the cell membrane integrity was obviously damaged(P<0.01).After exposure to 100 μmol·L-1 RUT for 24 h, the phosphorylation of ERK, JNK, STAT3 and p38 significantly increased(P<0.01, P<0.05), but there was no significant change in total protein level.The expression of c-Jun and c-Fos was up-regulated at 3 h(P<0.01), and at 3h time point, the phosphorylation of NF-κB p65 significantly increased(P<0.01), but nuclear translocation was not significant.Conclusions Under certain conditions, RUT shows cytotoxicity on HepG2 cells, and its toxic mechanism is mainly related to injury caused by oxidative stress and inflammatory response.
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@#【Objective】ThisstudyaimstoinvestigatewhetherrutecarpinehasaneffectoncalcificationofVSMCand itsunderlyingmechanism.【Methods】InvitromodelofratVSMCcalcificationwasusedinthisstudy.Rutecarpineat differentconcentrationswasusedtotreatculturedratVSMC.Theexpressionof Runx2，BMP2 and Osterix wasanalyzed byqRT-PCRandmineraldepositionwasdetectedbyalizarinredstaining.Inaddition，weexaminedtheeffectofrutecar⁃ pineonSirtuin-1（Sirt1）expressioninVSMCandtheeffectofSirt1inhibitoronVSMCcalcification.【Results】Alizarinred stainingandcalciumcontentassayshowedthatrutecarpineatdifferentconcentrationssignificantlyreducedcalcificationof ratVSMCinducedbyhighphosphorusandhighcalcium（136±10，75±6，52±6，31±5.29，P<0.05）.Usageofrutecar⁃ pinedecreasedtheactivityofALP，anosteogenicdifferentiationmolecularmarker，anddown-regulatedtheexpressionof Runx2（2.85±0.25，1.75±0.18，1.62±0.13，1.36±0.16，P <0.05），BMP2（3.2±0.32，1.85±0.17，1.65±0.15，1.43± 0.12，P<0.05）andOsterix（2.60±0.27，1.82±0.16，1.55±0.15，1.36±0.17，P<0.05），suggestingthatrutecarpineinhib⁃ ited osteogenic differentiation of VSMC. In addition，high phosphorus and high calcium down-regulated the expres⁃ sionofSirt1inVSMC.qRT-PCRandwesternblotanalysisconfirmedthatrutecarpineup-regulatedtheexpressionof Sirt1atbothmRNA（0.35±0.06，0.75±0.11；0.22±0.08，0.87±0.13，P <0.05）andproteinlevels（0.38±0.09，0.71±0.13，P<0.05）.QuantificationofcalciumcontentanalysisshowedinhibitionofSirt1byEX-527blockedtheinhibitory effectofrutecarpineonVSMCcalcification（138±13，36±7，87±8，P<0.05） .【Conclusion】Wedemonstratethatrutecar⁃ pineattenuatesVSMCcalcificationviaup-regulationofSirt1.
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AIM: To evaluate effects of rutecarpine on the movements of gastrointestinal tract in experimental animals. METHODS: In mice, the accelerated movement model of intestinal transit was induced by neostigmine, and metoclopramide or apoplon was applied to induce the accelerated gastric emptying movements. Acetylcholine or histamine was used to induce the contractions occurring in the isolated ileum from guinea pigs. RESULTS: Rutecarpine inhibited normal intestinal transit and demonstrated more effective suppression on the accelerated movement induced by neostigmine in mice; meto- clopramide and apoplon induced-accelerated gastric emptying movements were also significantly inhibited by rutecarpine in a dose-dependent manner. Meanwhile rutecarpine significantly inhibited the isolated ileum contractions induced by acetylcholine or histamine. CONCLUSION: Rutecarpine is an effective inhibitor to intestinal motility and this activity is probably mediated by its antagonistic effects on the cholinergic nerve or its responsible modulations.