Li-Ru-Kang (LRK), a formula of eight traditional Chinese medicines (TCM), has been used to treat hyperplasia of mammary gland (HMG) in TCM clinics. However, how LRK works in HMG patients is unclear. To explore the possible mechanisms of LRK against HMG, the network pharmacology was used to screen the potential targets and possible pathways that involved in LRK treated HMG. Rat HMG model induced by estrogen and progesterone was used to further verify the effects of the key molecules of LRK selected from the enriched pathways on HMG. Nipple heights and diameters were measured and uterus index was calculated. The histopathological changes of mammary gland tissue were detected by hematoxylin-eosin (H&E) staining. Western blot was used to detect the phosphorylation of ERK, JNK, and P38. And immunohistochemistry staining was performed to evaluate the levels of estrogen receptor α (ERα), progesterone receptor (PR), nuclear factor-(NF-)κB (p65), interleukin-1β (IL-1β), tumor necrosis factor α (TNF-α), cyclooxygenases 2 (COX-2), inducible nitric oxide synthase (iNOS), 8-hydroxy-2'deoxyguanosine (8-OHdG), and nitrotyrosine (NT). Our results indicate that LRK treatment rescues significantly nipples height and diameter, decreases uterus index and ameliorates HMG. LRK treatment also markedly attenuates the over-expression of IL-1β, TNF-α, COX-2, and iNOS, and suppressed the formation of 8-OHdG and NT. Furthermore, LRK treatment significantly inhibits the phosphorylation of JNK, ERK, and p38 and expression of NF-κB (p65), interestingly, LRK treatment has no effect on the expression of ERα and PR. Our data suggest that the LRK treatment protects the mammary glands from the damage of oxidative stress and inflammation induced by estrogen and progesterone, via suppresses of MAPK/NF-κB signaling pathways without affecting on the expression of ERα and PR.

Background: Li-Ru-Kang (LRK) has been used in the treatment of hyperplasia of mammary glands (HMG) for several decades and can effectively improve clinical symptoms. This study aims to investigate the mechanism by which LRK intervenes in HMG based on an integrated approach that combines metabolomics and network pharmacology analyses. Methods: The effects of LRK on HMG induced by estrogen-progesterone in rats were evaluated by analyzing the morphological and pathological characteristics of breast tissues. Moreover, UPLC-QTOF/MS was performed to explore specific metabolites potentially affecting the pathological process of HMG and the effects of LRK. Pathway analysis was conducted with a combination of metabolomics and network pharmacology analyses to illustrate the pathways and network of LRK-treated HMG. Results: Li-Ru-Kang significantly improved the morphological and pathological characteristics of breast tissues. Metabolomics analyses showed that the therapeutic effect of LRK was mainly associated with the regulation of 10 metabolites, including prostaglandin E2, phosphatidylcholine, leukotriene B4, and phosphatidylserine. Pathway analysis indicated that the metabolites were related to arachidonic acid metabolism, glycerophospholipid metabolism and linoleic acid metabolism. Moreover, principal component analysis showed that the metabolites in the model group were clearly classified, whereas the metabolites in the LRK group were between those in the normal and model groups but closer to those in the normal group. This finding indicated that these metabolites may be responsible for the effects of LRK. The therapeutic effect of LRK on HMG was possibly related to the regulation of 10 specific metabolites. In addition, we further verified the expression of protein kinase C alpha (PKCα), a key target predicted by network pharmacology analysis, and showed that LRK could significantly improve the expression of PKCα. Conclusion: Our study successfully explained the modulatory properties of LRK treatment on HMG using metabolomics and network pharmacology analyses. This systematic method can provide methodological support for further understanding the complex mechanism underlying HMG and possible traditional Chinese medicine (TCM) active ingredients for the treatment of HMG.

Metabolic reprogramming is a cancer hallmark. Although the reprogramming of central carbon has been well documented, the role of sulfur metabolism has been largely overlooked. Additionally, the effects of sulfur are sometimes contradictory in tumorigenesis. In this study, we aimed to investigate the gene expression profile in hepatocellular carcinoma (HCC) and the effects of reactive sulfur species (RSS) on HCC tumor cells. Furthermore, the cell imaging technology was applied to discover some potential anti-cancer compounds. Gene Set Enrichment Analysis (GSEA) of Gene Expression Omnibus (GEO) dataset (GSE102083) revealed that sulfur amino acid-related metabolism and vitamin B6 binding activity in HCC tissues were downregulated. Calculation of the interaction network identified nine hub genes, among which eight were validated by differential expression and survival analysis in the TCGA_LIHC cohort, and two (CSE and CBS) had the highest enrichment degree. The metabolomics analysis suggested that the hub genes were associated with RSS metabolism including H2S, H2S2, cystine, cysteine, homocysteine, cystathionine, and methionine. The cell viability assay demonstrated that H2S2 had significant anti-cancer effects in HCC SNU398 tumor cells. The cell imaging assay showed that treatment with H2S2 remarkably increased intracellular sulfane sulfur content. On this basis, the anti-cancer activity of some other sulfane sulfur compounds, such as DATS and DADS, was further verified. Lastly, according to the fact that HCC tumor cells preferentially take in cystine due to high expression of SLC7A11 (a cystine/glutamate transporter), persulfided cysteine precursor (PSCP) was tested for its sulfane sulfur release capability and found to selectively inhibit HCC tumor cell viability. Collectively, this study uncovered sulfur metabolism in HCC was reprogrammed, and provided a potential therapeutic strategy for HCC by donating sulfane sulfur.

Objective: San-Cao granule (SCG), a traditional Chinese herb formula, has been used for treating autoimmune hepatitis (AIH) in our clinics for a long time. However, its active ingredients and mechanisms of action were still unknown due to its complicated chemical compositions. In the present study, the pharmacological study of SCG on acute liver injury induced by Concanavalin A (Con A) was performed to provide a scientific evidence for SCG against liver injury. Methods: In order to screen active components and predicate mechanisms of action, an "ingredients-target-disease" interaction network was constructed by network pharmacology. Then, the pharmacological study was performed to evaluate the therapeutic effect and the underlying mechanisms of SCG on Con A-induced liver injury in mice. Results: This research demonstrated the pharmacological effect of SCG on Con A-induced liver injury, which was through improving the liver function, relieving the pathological changes of liver tissue, decreasing the level of pro-inflammatory cytokines, and thus balancing the pro- and anti-inflammatory cytokines. And the anti-inflammatory of SCG may advantage over the ursodeoxycholic acid (UDCA). Network pharmacology analysis revealed that the pharmacological effect of SCG might be related to its active ingredients of taraxanthin, dihydrotanshinone I, isotanshinone I, γ-sitosterol, 3β-acetyl-20,25-epoxydammarane-24α, and δ-7-stigmastenol. The hepatoprotective effect of SCG was reflected by suppressing Con A-induced apoptosis which was mediated by TRAIL and FASL. Conclusion: The combination of network pharmacology and experimental data has revealed the anti-apoptotic effect of SCG against Con A-induced liver injury.

Backgrounds: Salsolinol (SAL), a plant-based isoquinoline alkaloid, was initially isolated from Aconiti Lateralis Radix Praeparata (ALRP) and identified as the active cardiotonic component of ALRP. This study was aimed to explore the therapeutic effect and mechanism by which SAL attenuates doxorubicin (DOX)-induced chronic heart failure (CHF) in rats and improves mitochondrial function in H9c2 cardiomyocytes. Methods: Rats were intraperitoneally injected with DOX to establish CHF model. Therapeutic effects of SAL on hemodynamic parameters, serum indices, and the histopathology of the heart were analyzed in vivo. Moreover, H9c2 cardiomyocytes were pretreated with SAL for 2 h before DOX treatment in all procedures in vitro. Cell viability, cardiomyocyte morphology, proliferation, and mitochondrial function were detected by a high-content screening (HCS) assay. In addition, a Seahorse Extracellular Flux (XFp) analyzer was used to evaluate the cell energy respiratory and energy metabolism function. To further investigate the potential mechanism of SAL, relative mRNA and protein expression of key enzymes in the tricarboxylic acid cycle in vivo and mitochondrial calcium uniporter (MCU) signaling pathway-related molecules in vitro were detected. Results: The present data demonstrated the pharmacological effect of SAL on DOX-induced CHF, which was through ameliorating heart function, downregulating serum levels of myocardial injury markers, alleviating histological injury to the heart, increasing the relative mRNA expression levels of key enzymes downstream of the tricarboxylic acid cycle in vivo, and thus enhancing myocardial energy metabolism. In addition, SAL had effects on increasing cell viability, ameliorating DOX-induced mitochondrial dysfunction, and increasing mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in H9c2 cardiomyocyte. Moreover, we found that SAL might have an effect on improving mitochondrial respiratory function and energy metabolism via inhibiting excessive activation of MCU pathway in H9c2 cells. However, the protective effect could be ameliorated by ruthenium red (an MCU inhibitor) and abrogated by spermine (an MCU activator) in vitro. Conclusion: The therapeutic effects of SAL on CHF are possibly related to ameliorating cardiomyocyte function resulting in promotion of mitochondrial respiratory and energy metabolism. Furthermore, the potential mechanism might be related to downregulating MCU pathway. These findings may provide a potential therapy for CHF.

The contribution of the metabolites of linezolid to the associated myelosuppression is unknown in patients who are renal impairment. In this research, the purpose of our experiment was to explore and develop a quick and robust ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for the determination of linezolid and its metabolite PNU-142300 in human serum simultaneously. The analytes were prepared using a simple and convenient approach with acetonitrile for protein crash, and then separated from the matrix on a Waters Acquity Ultra performance liquid chromatography (UPLC) BEH C18 (2.1 mm × 50 mm, 1.7 μm) column in a program of gradient elution, where the mobile phase was consisted of water with 0.1% formic acid and acetonitrile, and was placed at 0.40 ml/min flow rate. Multiple reaction monitoring (MRM) was employed and conducted for UPLC-MS/MS detection with ion transitions at m/z 338.01 → 296.03 for linezolid, m/z 369.96 → 327.98 for PNU-142300 and m/z 370.98 → 342.99 for tedizolid (Internal standard, IS), respectively. This method had good linearity respectively in the calibration range of 0.01-20 μg/ml for linezolid, and 0.05-100 μg/ml for PNU-142300. In the intra- and inter-day, the precision of linezolid and PNU-142300 was below 14.2%, and the accuracy in this method was determined to be from -9.7 to 12.8%. In addition, recovery and matrix effect of the analytes were all found to be acceptable, and the analytes during the assay and storage in serum samples were observed to be stable. The novel optimized UPLC-MS/MS assay was also successfully employed to determine the concentration levels of linezolid and PNU-142300 in human serum. The results showed that linezolid-associated myelosuppression occurs more frequently in patients with renal insufficiency, and the metabolite-to-parent concentration ratio of PNU-142300 is predicted to reduce this toxicity of myelosuppression.

DT-13, a saponin monomer 13 from the dwarf lilyturf tuber, was reported to exhibit anti-inflammatory, hepatoprotective, cardioprotective as well as antitumor activities in a number of tumor cells. Prostate cancer is the second leading cause of cancer death in males, discovery of novel antitumor drug for therapy of prostate cancer is expected. Aiming to evaluate whether DT-13 could become a candidate to treat prostate cancer, we recently investigated the antitumor effect of DT-13 on human prostate cancer cells and the underlying mechanism. DT-13 was found to effectively inhibit proliferation and metastasis of prostate cancer PC3 and DU145 cell lines in a dose-dependent manner. Treatment by DT-13 resulted in a mitochondria-mediated apoptosis, which was accompanied by the chromatin condensation and nuclear shrinkage in the prostate cancer cells. Moreover, DT-13 caused remarkable upregulation of Bax, Bad, Cytochrome C, cleaved -caspase 3, -caspase 9 and -PARP, in contrast to the downregulation of Bcl-2. Nevertheless, no obvious change in intracellular ROS level was observed after DT-13 treatment. We further demonstrated that DT-13 could inhibit PC3 cell metastasis in which suppression of Integrinβ1 and MMP2/9 might be involved. Western blot analysis indicated DT-13 significantly decreased the phosphorylation of PDK1, Akt, mTOR as well as p70S6K, suggesting the pro-apoptotic and anti-metastatic effects of DT-13 on prostate cancer cells might be attributed to the blockade of PI3K/Akt pathway. Collectively, our findings suggest DT-13 is worthy of further investigation as a drug candidate for the treatment of prostate cancer.

Digoxin is widely used to treat heart failure. Epidemiological studies suggested it might be used as an anticancer drug or sensitizing agent for cancer therapy. Adriamycin is a well-known anticancer drug, but often causes cardiotoxicity which limits its use. We recently investigated the anticancer effects of digoxin alone or in combination with adriamycin on human non-small cell lung cancer in vitro and in vivo. Digoxin reduced the viability of A549 and H1299 cells in vitro, increased DNA damage by promoting ROS generation and inhibiting both DNA double strand break (DSB) and single strand break (SSB) repair. Combination with adriamycin showed synergistic antiproliferative effects at the ratios of 1/2IC50DIG:IC50ADR and IC50DIG:IC50ADR on A549 and H1299 cells, respectively. In vivo, digoxin potently inhibited A549 growth in both zebrafish and nude mouse xenograft model. Co-treatment with adriamycin not only enhanced the antitumor efficacy, but also reduced the cardiotoxicity. Our findings suggest that digoxin has the potential to be applied as an antitumor drug via inhibiting both DNA DSB and SSB repair, and combination with adriamycin for therapy of human non-small cell lung cancer is reasonable.