Integrating serum pharmacochemistry and network pharmacology to explore the molecular mechanisms of Acanthopanax senticosus (Rupr. & Maxim.) Harms on attenuating doxorubicin-induced myocardial injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Acanthopanax senticosus (Rupr. & Maxim.) Harms (AS), also known as Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. or Siberian ginseng, has a rich history of use as an adaptogen, a substance believed to increase the body's resistance to stress, fatigue, and infectious diseases. As a traditional Chinese medicine, AS is popular for its cardioprotective effects which can protect the cardiovascular system from hazardous conditions. Doxorubicin (DOX), on the other hand, is a first-line chemotherapeutic agent against a variety of cancers, including breast cancer, lung cancer, gastric cancer, and leukemia, etc. Despite its effectiveness, the clinical use of DOX is limited by its side effects, the most serious of which is cardiotoxicity. Considering AS could be applied as an adjuvant to anticancer agents, the combination of AS and DOX might exert synergistic effects on certain malignancies with mitigated cardiotoxicity. Given this, it is necessary and meaningful to confirm whether AS would neutralize the DOX-induced cardiotoxicity and its underlying molecular mechanisms. AIM OF THE STUDY: This paper aims to validate the cardioprotective effects of AS against DOX-induced myocardial injury (MI) while deciphering the molecular mechanisms underlying such effects. MATERIALS AND METHODS: Firstly, the cardioprotective effects of AS against DOX-induced MI were confirmed both in vitro and in vivo. Secondly, serum pharmacochemistry and network pharmacology were orchestrated to explore the in vivo active compounds of AS and predict their ways of functioning in the treatment of DOX-induced MI. Finally, the predicted mechanisms were validated by Western blot analysis during in vivo experiments. RESULTS: The results demonstrated that AS possessed excellent antioxidative ability, and could alleviate the apoptosis of H9C2 cells and the damage to mitochondria induced by DOX. In vivo experiments indicated that AS could restore the conduction abnormalities and ameliorate histopathological changes according to the electrocardiogram and cardiac morphology. Meanwhile, it markedly downregulated the inflammatory factors (TNF-α, IL-6, and IL-1ß), decreased plasma ALT, AST, LDH, CK, CK-MB, and MDA levels, as well as increased SOD and GSH levels compared to the model group, which collectively substantiate the effectiveness of AS. Afterward, 14 compounds were identified from different batches of AS-dosed serum and selected for mechanism prediction through HPLC-HRMS analysis and network pharmacology. Consequently, the MAPKs and caspase cascade were confirmed as primary targets among which the interplay between the JNK/Caspase 3 feedback loop and the phosphorylation of ERK1/2 were highlighted. CONCLUSIONS: In conclusion, the integrated approach employed in this paper illuminated the molecular mechanism of AS against DOX-induced MI, whilst providing a valuable strategy to elucidate the therapeutic effects of complicated TCM systems more reliably and efficiently.

Bioconversion of Ginsenoside Rf into Its Hydrated Derivative with Elevated Anti-inflammatory Effect by a Highly Specific Biocatalytic System of Cordyceps Sinensis.

The presence of 25-OH moiety has been proved to enhance the bioactivity of dammarane saponins in many cases. However, such modification by previous strategies had compromised yield and purity of target products. Herein ginsenoside Rf was specifically transformed into 25-OH-(20S)-Rf with a conversion rate of 88.03 % by a Cordyceps Sinensis-mediated biocatalytic system. The formulation of 25-OH-(20S)-Rf was calculated by HRMS, whilst its structure was validated by 1 H-NMR, 13 C-NMR, HSQC, and HMBC analysis. Time-course experiments unveiled straightforward hydration of the double bond on Rf with undetectable side reactions and maximum production of 25-OH-(20S)-Rf on the 6th day, which collectively suggested the suitable timing of harvesting this target compound. In vitro bioassay of (20S)-Rf and 25-OH-(20S)-Rf against lipopolysaccharide-induced macrophages indicated a significant boost of anti-inflammatory effects after the C24-C25 double bond was hydrated. Therefore, the biocatalytic system in this article could be leveraged to deal with macrophage-mediated inflammation under defined circumstances.

Cordyceps sinensis-mediated biotransformation of notoginsenoside R1 into 25-OH-20(S/R)-R2 with elevated cardioprotective effect against DOX-induced cell injury.

Notoginsenoside R1 is a dammarane saponin in Panax notoginseng with promising cardioprotective effects. The bioactivity-structure relationship of such saponins suggested that the presence of a hydroxyl group at C25 could elevate its performance. To fulfill that goal, bioconversion of notoginsenoside R1 was mediated by a biocatalytic system of Cordyceps sinensis that had successfully produced multiple 25-OH derivatives from ginsenoside Re and Rg1. The major metabolic products of notoginsenoside R1 were identified as 25-OH-20(S/R)-R2 via the techniques of HRMS, 13C-NMR, 1H-NMR, HSQC and HMBC. Time-course experiments were designed to monitor the reaction process, establishing a biocatalytic pathway of "R1→20(S/R)-R2→25-OH-20(S/R)-R2". The bioconversion rate of these 25-OH derivatives added up to 69.87% which greatly precedes the previous report. Afterwards, the effect of these biocatalytic products against doxorubicin-induced cardiotoxicity was evaluated, indicating a significant increase in efficacy after the hydration of the C24-C25 double bond on the dammarane skeleton. In conclusion, the biocatalytic system employed in this paper is able to harvest 25-OH-20(S/R)-R2 in high yield from notoginsenoside R1, which will provide lead compounds or drug candidates to alleviate myocardial injury caused by doxorubicin.

Interaction Networks Converging on Immunosuppressive Roles of Granzyme B: Special Niches Within the Tumor Microenvironment.

Granzyme B is a renowned effector molecule primarily utilized by CTLs and NK cells against ill-defined and/or transformed cells during immunosurveillance. The overall expression of granzyme B within tumor microenvironment has been well-established as a prognostic marker indicative of priming immunity for a long time. Until recent years, increasing immunosuppressive effects of granzyme B are unveiled in the setting of different immunological context. The accumulative evidence confounded the roles of granzyme B in immune responses, thereby arousing great interests in characterizing detailed feature of granzyme B-positive niche. In this paper, the granzyme B-related regulatory effects of major suppressor cells as well as the tumor microenvironment that defines such functionalities were longitudinally summarized and discussed. Multiplex networks were built upon the interactions among different transcriptional factors, cytokines, and chemokines that regarded to the initiation and regulation of granzyme B-mediated immunosuppression. The conclusions and prospect may facilitate better interpretations of the clinical significance of granzyme B, guiding the rational development of therapeutic regimen and diagnostic probes for anti-tumor purposes.

Highly regioselective biotransformation of ginsenoside Rg1 to 25-OH derivatives of 20(S/R)-Rh1 by Cordyceps Sinensis.

25-OH ginsenosides are potent and rare prodrugs in natural sources. However current strategies for such modification always end up in undesirable side products and unsatisfied yield that hinders them from further applications. Herein, ginsenoside Rg1 was thoroughly converted into 20(S/R)-Rh1 and 25-OH-20(S/R)-Rh1 by Cordyceps Sinensis in an optimum medium. The chemical correctness of either 25-OH-20(S/R)-Rh1 epimers was validated by LC-IT-TOF-MSn and 13C NMR spectrometry. The biocatalytic pathway was established as Rg1 â†’ 20(S/R)-Rh1 â†’ 25-OH-20(S/R)-Rh1. The molar bioconversion rate for total 25-OH-20(S/R)-Rh1 was calculated to be 82.5%, of which S-configuration accounted for 43.2% while R-configuration 39.3%. These two 25-OH derivatives are direct hydration products from 20(S/R)-Rh1 without other side metabolites, suggesting this is a highly regioselective process. In conclusion, this biocatalytic system could be harnessed to facilitate the preparation of diversified 25-OH ginsenosides with high yields of the target compound and simple chemical background in the reaction mixture.

Adsorption and desorption characteristics of ginsenosides from Panax ginseng C. A. Meyer on middle-pressure chromatogram isolated gels.

Four types of middle-pressure chromatogram isolated gels are evaluated for adsorption or desorption characteristics of ginsenosides from Panax ginseng. Among them, SP207SS and SP2MGS were selected for dynamic investigations based on their static adsorption or desorption capacity of total ginsenoside. Their adsorption kinetics was better explained by pseudosecond-order model and isotherms were preferably fitted to Langmuir model. Dynamic breakthrough experiments indicated an optimum sample loading speed of 4 bed volume/h for either SP207SS or SP2MGS. Desorption speed was determined to be 2 bed volume/h according to desorption amount of total ginsenoside in their effluents. Eight ginsenosides were identified and quantified by high performance liquid chromatography-triple quadropole-mass spectrometry in total ginsenoside extract and different fractions during stepwise dynamic elution. For SP207SS, 27.62% of loaded ginsenosides was detected in 40% ethanol fraction, while 59.12% of them were found in 60% ethanol fraction. As on SP2MGS, the number went to 53.71 and 44.43%, respectively. Recovery rate of ginsenosides were calculated to 78.65% for SP207SS and 89.53% for SP2MGS, respectively. Intriguingly, content of Rg1 and Re in 40% ethanol fraction from SP207SS became 20.1 and 18.6 times higher than that in total ginsenoside extract by one-step elution, which could be leveraged for the facile enrichment of these two ginsenosides from natural sources.

Two common SNPs in pri-miR-125a alter the mature miRNA expression and associate with recurrent pregnancy loss in a Han-Chinese population.

Although there are plenty of evidence that single-nucleotide polymorphisms (SNPs) that fall within coding sequences of genes are involved in recurrent pregnancy loss (RPL), it is still unknown whether the polymorphisms in microRNAs (miRNAs) are related with RPL. In this study, we established this kind of association by confirming significant differences in genotype distribution of rs41275794 (P= 0.0005) and rs12976445 (P= 0.001) within the pri-miR-125a in 217 Han Chinese patients of RPL compared with 431 controls. Based on this observation, two-locus haplotypes were constructed and the A-T haplotype was found to be associated with an increased risk of RPL (OR=2.84, 95%C.I. 1.98-4.07, P=0.0000000057). Further analysis showed that the levels of pre- and mature- miR-125a were down-regulated in the cells transfected with the A-T haplotype, which was consistent with in vivo detection that the level of mature miR-125a was lower in 30 pregnant women with A-T haplotype than that with G-C haplotype. During in vitro RNA processing assays, we found a similar decrease in the amount of pre-miR-125a and decline in binding capacity of nuclear factors to pri-miR-125a with A-T haplotype. More importantly, the reduction in miR-125a, as a consequence of A-T haplotype, further led to less efficient inhibition of target genes, LIFR and ERBB2, which play important roles in the embryo implantation and decidualization. Thus, our data collectively suggest that two common polymorphisms in pre-miR-125a might contribute to the genetic predisposition to RPL by disrupting the production of miR-125a, which consequently interfered in the expression and function of target genes of miR-125a.

MiR-125b expression affects the proliferation and apoptosis of human glioma cells by targeting Bmf.

BACKGROUND: MicroRNAs (miRNAs) are small noncoding RNAs whose function as modulators of gene expression is crucial for the proper control of cell growth. Although many microRNAs were found to express in central nervous system (CNS), the role of the regulatory networks in which they are involved and their function in the pathological process of nerve cells are only just emerging. In the present study, the possible mechanisms by which one neuronal miRNAs, miR-125b, affected the growth of nervous cells were investigated using in vitro cell line model. METHODS: The expression pattern of miR-125b in ATRA-treated human glioma cell lines was detected by Northern blotting and in situ localization. The effect of miR-125b on the proliferation and apoptosis of human glioma cells was analyzed by MTS assay, TUNEL and Flow cytometry analysis. In addition, the identification of target gene of miR-125b was studied by dual-luciferase activity assay and Immunoblot Analysis. RESULTS: We found differential expression of miR-125b in 1.0 microM all-trans-retinoic acid (ATRA)-treated human glioma cell lines. Up-regulation of miR-125b partially restored cell viability and inhibited cell apoptosis in U343 cells treated by ATRA. Down-regulation of miR-125b decreased human glioma cells proliferation and enhanced the sensitivity of human glioma cells to ATRA-induced apoptosis. In addition, we found an inverse relationship between the expression of miR-125b and the cell apoptosis-related protein Bcl-2 modifying factor (Bmf), and miR-125b can interact with 3'-untranslated region (UTR) of Bmf. CONCLUSION: These findings indicate that overexpression of miR-125b promotes human glioma cell proliferation and inhibits ATRA-induced cell apoptosis and low expression of miR-125b sensitizes cells to ATRA-induced apoptosis. BMF may play an important role in the process of miR-125b influencing cell apoptosis.