OBJECTIVE Neocryptotanshinone(NCTS)is a natural product extracted from traditional Chinese herb Salvia miltiorrhiza Bunge.Previous studies have demonstrated the anti-inflammatory of NCTS in lipopolysaccharide(LPS)-sti...OBJECTIVE Neocryptotanshinone(NCTS)is a natural product extracted from traditional Chinese herb Salvia miltiorrhiza Bunge.Previous studies have demonstrated the anti-inflammatory of NCTS in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW 264.7).However,the protective effect and mechanism of NCTS in cardiomy⁃ocytes are still undefined.This study is to investigate whether NCTS exerts its cardioprotective effect against hypoxia/re⁃oxygenation(H/R)-induced H9C2 cell injury.METHODS The model of H/R injury was established through hypoxia for 8 h and reoxygenation for 12 h in H9C2 cardiomyocytes of rats.Cultured cardiomyocytes were randomly divided into four groups,control group,H/R group,H/R+NCTS pretreated group(1,2,5 and 10μmol·L^-1),and H/R+NCTS+HX531(an RXRαantagonist,2μmol·L^-1)co-treated group.The cell viability was measured by Cell Counting Kit-8,Hoechst33258 staining was used to observe the morphology of apoptotic changes.Mitochondrial membrane potential was detected by JC-1 fluorescent probe,and protein expressions of RXRα,Bcl-2,Bax,caspase-3 and cleaved caspase-3 with Western blotting.RESULTS Compared with control group,the cell viability in model group was decreased(P<0.05).After treated with NCTS in different concentrations,the CCK8 results showed that NCTS in 2μmol·L^-1 had protective effects.Result of Hoechst33258 staining suggested that the apoptosis was notably increased in model group(P<0.05),Meanwhile,the JC-1 results showed that the mitochondrial membrane potential of the model group decreased which was consistent with previous study.impressively,NCTS could restore the mitochondrial membrane potential as well as apoptosis.Fur⁃ther western blot experiments showed that NCTS treat could upregulate Bcl-2 protein,and downregulate the levels of Bax and cleaved caspase-3/caspase-3 ratio.Since RXRαis a critical upstreaming proteins which can directly mediate the apoptosis,we then determined the effect of NCTS on it.Intriguingly,RXRαwas notably activated by NCTS,while the HX531,the antagonist of RXRα,could abolished NCTS'effect when co-treated with NCTS.CONCLUSION NCTS in 2μmol·L^-1 was effective to protect H9C2 cell from H/R-induced cell injury through RXRα-mediated mitochondria apop⁃tosis.Current results provide possible drugs for the treatment of ischemic cardiomyopathy.展开更多
Neocryptotanshinone(NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide(LPS)-stimulated mouse...Neocryptotanshinone(NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α(TNFα), interleukin-6(IL-6) and interleukin-1β(IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide(NO)production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase(iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2(COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation.In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.展开更多
Danshen,the dried roots and rhizomes of Salvia miltiorrhiza Bunge(S.miltiorrhiza),is widely used in the treatment of cardiovascular and cerebrovascular diseases.Tanshinones,the bioactive compounds from Danshen,exhibit...Danshen,the dried roots and rhizomes of Salvia miltiorrhiza Bunge(S.miltiorrhiza),is widely used in the treatment of cardiovascular and cerebrovascular diseases.Tanshinones,the bioactive compounds from Danshen,exhibit a wide spectrum of pharmacological properties,suggesting their potential for future therapeutic applications.Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized,most of which belong to the CYP76 and CYP71 families.In this study,CYP81C16,a member of the CYP71 clan,was identified in S.miltiorrhiza.An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones,namely neocryptotanshinone,deoxyneocryptotanshinone,and danshenxinkuns A and B.SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%.Kinetic evaluations and in vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates.The overexpression of SmCYP81C16 promoted the accumulation of(iso)tanshinone in hairy root lines.The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones,either through microbial or plant metabolic engineering.展开更多
基金National Natural Science Foundation of China(81822049and 81673712)
文摘OBJECTIVE Neocryptotanshinone(NCTS)is a natural product extracted from traditional Chinese herb Salvia miltiorrhiza Bunge.Previous studies have demonstrated the anti-inflammatory of NCTS in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW 264.7).However,the protective effect and mechanism of NCTS in cardiomy⁃ocytes are still undefined.This study is to investigate whether NCTS exerts its cardioprotective effect against hypoxia/re⁃oxygenation(H/R)-induced H9C2 cell injury.METHODS The model of H/R injury was established through hypoxia for 8 h and reoxygenation for 12 h in H9C2 cardiomyocytes of rats.Cultured cardiomyocytes were randomly divided into four groups,control group,H/R group,H/R+NCTS pretreated group(1,2,5 and 10μmol·L^-1),and H/R+NCTS+HX531(an RXRαantagonist,2μmol·L^-1)co-treated group.The cell viability was measured by Cell Counting Kit-8,Hoechst33258 staining was used to observe the morphology of apoptotic changes.Mitochondrial membrane potential was detected by JC-1 fluorescent probe,and protein expressions of RXRα,Bcl-2,Bax,caspase-3 and cleaved caspase-3 with Western blotting.RESULTS Compared with control group,the cell viability in model group was decreased(P<0.05).After treated with NCTS in different concentrations,the CCK8 results showed that NCTS in 2μmol·L^-1 had protective effects.Result of Hoechst33258 staining suggested that the apoptosis was notably increased in model group(P<0.05),Meanwhile,the JC-1 results showed that the mitochondrial membrane potential of the model group decreased which was consistent with previous study.impressively,NCTS could restore the mitochondrial membrane potential as well as apoptosis.Fur⁃ther western blot experiments showed that NCTS treat could upregulate Bcl-2 protein,and downregulate the levels of Bax and cleaved caspase-3/caspase-3 ratio.Since RXRαis a critical upstreaming proteins which can directly mediate the apoptosis,we then determined the effect of NCTS on it.Intriguingly,RXRαwas notably activated by NCTS,while the HX531,the antagonist of RXRα,could abolished NCTS'effect when co-treated with NCTS.CONCLUSION NCTS in 2μmol·L^-1 was effective to protect H9C2 cell from H/R-induced cell injury through RXRα-mediated mitochondria apop⁃tosis.Current results provide possible drugs for the treatment of ischemic cardiomyopathy.
基金supported by the Science and Technology Development Fund of Macao Special Administrative Region (No. 021/2012/A1)the Research Fund of University of Macao (No. MRG007/CXP/2013/ICMS)
文摘Neocryptotanshinone(NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α(TNFα), interleukin-6(IL-6) and interleukin-1β(IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide(NO)production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase(iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2(COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation.In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.
基金This work was supported by the National Key R&D Program of China(Nos.2020YFA0908000,2018YFA0900600)the National Natural Science Foundation of China(Nos.82003904,81822046)+4 种基金the Fundamental Research Funds for the Central public welfare research institutes(No.ZZ13-YQ-083)a Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences(No.CI2021A04110)Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(No.ZYYCXTD-D-202005)a key project at central government level(the ability to establish sustainable use of valuable Chinese medicine resources2060302,China).
文摘Danshen,the dried roots and rhizomes of Salvia miltiorrhiza Bunge(S.miltiorrhiza),is widely used in the treatment of cardiovascular and cerebrovascular diseases.Tanshinones,the bioactive compounds from Danshen,exhibit a wide spectrum of pharmacological properties,suggesting their potential for future therapeutic applications.Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized,most of which belong to the CYP76 and CYP71 families.In this study,CYP81C16,a member of the CYP71 clan,was identified in S.miltiorrhiza.An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones,namely neocryptotanshinone,deoxyneocryptotanshinone,and danshenxinkuns A and B.SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%.Kinetic evaluations and in vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates.The overexpression of SmCYP81C16 promoted the accumulation of(iso)tanshinone in hairy root lines.The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones,either through microbial or plant metabolic engineering.
