RESUMO
Akebia saponin D (ASD) is derived from the Dipsacus asper Wall. ex Henry, which is a traditional Chinese medicine commonly used to treat rheumatic arthritis (RA). However, the in-depth mechanism of the anti-inflammatory effect of ASD is still unclear. This study aimed to preliminarily explore the anti-inflammatory effect of ASD and the underlying mechanisms from the perspective of DNA methylation and inflammation-related pathways. We found that ASD significantly reduced the production of multiple inflammatory mediators, including nitric oxide (NO) and prostaglandin E2 (PGE2), in LPS-induced RAW264.7 cells. The expression of DNA methyltransferase (DNMT) 3b and inducible nitric oxide synthase (iNOS) was also obviously inhibited by the ASD treatment. The protein and mRNA levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also significantly inhibited by ASD. ASD inhibited the macrophage M1 phenotype, inhibited the high level of DNMT3b, and downregulated the signal transducer and activator of the transcription 3 (STAT3) pathway to exert its anti-inflammatory activity. Furthermore, DNMT3b siRNA and Nrf2 siRNA significantly promoted the anti-inflammatory effect of ASD. Our study demonstrates for the first time that ASD inhibits the IL-6-STAT3-DNMT3b axis and activates the nuclear factor-E2-related factor 2 (Nrf2) signaling pathway to achieve its inhibitory effect on inflammatory reactions.
Assuntos
Interleucina-6 , Fator 2 Relacionado a NF-E2 , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , DNA/uso terapêutico , Dinoprostona/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Metiltransferases/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro , RNA Interferente Pequeno/uso terapêutico , Fator de Transcrição STAT3 , Saponinas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Objective: To clone and screen the stable internal reference genes from Dipsacus asper for qRT-PCR analysis correction, so as to provide a preliminary basis for future research on expression analysis and regulation mechanism of D. asper functional genes. Methods: The internal reference genes of Actin, Tubulin and GAPDH gene families were screened and cloned from D. asper transcriptome database. The D. asper plants from different origins, different tissues and different developmental stages were used to obtain expression information of each gene by qRT-PCR. The expression stability of each gene was analyzed by geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, and the best genes were synthetically evaluated and screened. Results: Ten core fragments for candidate internal reference genes were cloned, belonging to three gene families: Actin, Tubulin and GAPDH, with high homology among them. The results of stability analysis showed that the expression of DaACT103 was stable and relatively high in different regions and tissues, while the expression of DaTUB5 was stable and relatively low in different developmental stages. Conclusion:s DaACT103 and DaTUB5 are suitable as the internal reference genes for D. asper. DaACT103 is used as the internal reference gene with high abundances and DaACT105 is used as the internal reference gene with low abundances.
RESUMO
Objective: To study the genetic diversity of Dipsacus asper from different populations and provide a reference for the rational utilization of its germplasm. Methods: The genetic diversity of the 14 populations of D. asper was analyzed by SRAP molecular markers. Results: Ten pairs of primers produced 124 sites, among which 102 were polymorphic sites. The percentage of polymorphic loci (PPL) was 82.26%. The Nei's genetic diversity index (H) and the Shannon's information index (I) were 0.2800 and 0.4353, respectively. At the population level, PPL was 53.92%, H was 0.1212-0.2440, and I was 0.1796-0.3611. The genetic diversity values of the five populations were relatively high, and the populations had the characteristics of high altitude and microhabitat. Genetic differentiation coefficient (Gst) was 0.2930, gene flow (Nm) was 1.2064. Cluster analysis based on genetic similarity indicated that the 14 populations could be divided into three groups. Conclusion: The genetic diversity among the populations of D. asper was at relatively high level. The genetic variance of D. asper mainly existed within the populations. The high genetic diversity could be attributed to the geographical position (altitude) and climate, while geographic isolation (microhabitat) was another important factor for the genetic variance within the populations.
