Transcriptomic Analysis Under Drought and Salt Stress Provides Insight into Genes Putatively Involved in Ginsenoside Biosynthesis in Panax japonicus Meyer.

Panax japonicus Meyer, a perennial herb of the dicotyledonaceae family Araliaceae, is a rare folk traditional Chinese medicine, known as "the king of herbal medicine" in China. To understand the genes involved in secondary pathways under drought and salt stress, the transcriptomic analysis of P. japonicus is of vital importance. The transcriptome of underground rhizomes, stems, and leaves under drought and salt stress in P. japonicus were performed using the Illumina HiSeq platform. After de novo assembly of transcripts, expression profiling and identified differentially expressed genes (DEGs) were performed. Furthermore, putative functions of identified DEGs correlated with ginsenoside in P. japonicus were explored using Gene Ontology terms and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis. A total of 221,804 unigenes were obtained from the transcriptome of P. japonicus. The further analysis revealed that 10,839 unigenes were mapped to 91 KEGG pathways. Furthermore, a total of two metabolic pathways of P. japonicus in response to drought and salt stress related to triterpene saponin synthesis were screened. The sesquiterpene and triterpene metabolic pathways were annotated and finally putatively involved in ginsenoside content and correlation analysis of the expression of these genes were analyzed to identify four genes, ß-amyrin synthase, isoprene synthase, squalene epoxidase, and 1-deoxy-D-ketose-5-phosphate synthase, respectively. Our results paves the way for screening highly expressed genes and mining genes related to triterpenoid saponin synthesis. It also provides valuable references for the study of genes involved in ginsenoside biosynthesis and signal pathway of P. japonicus.

LMNB1 targets FOXD1 to promote progression of prostate cancer.

Forkhead box D1 (FOXD1) expression is upregulated in various types of human cancer. To the best of our knowledge, the roles of FOXD1 in prostate cancer (PC) remain largely unknown. The Cancer Genome Atlas dataset was used for the bioinformatics analysis of FOXD1 in PC. FOXD1 expression levels in normal immortalized human prostate epithelial cells (RWPE-1) and prostate cancer cells were detected by reverse transcription-quantitative PCR. PC cell viability was detected using Cell Counting Kit-8 assay. Transwell assays were performed to assess the migration and invasion of PC cells. Luciferase reporter gene assay was used to validate the association between FOXD1 and lamin (LMN)B1. LMNB1 is an important part of the cytoskeleton, which serves an important role in the process of tumor occurrence and development, regulating apoptosis and DNA repair. FOXD1 expression was upregulated in PC tissues, with its high expression being associated with clinical stage and survival in PC. Knockdown of FOXD1 inhibited viability, migration and invasion of PC cells. FOXD1 positively regulated LMNB1 expression. The effect of FOXD1 knockdown on PC cells was reversed by LMNB1 overexpression. In conclusion, FOXD1, positively regulated by LMNB1, served as an oncogene in PC and may be a potential biomarker and treatment target for PC.

Investigation of the mechanism of Prunella vulgaris in treatment of papillary thyroid carcinoma based on network pharmacology integrated molecular docking and experimental verification.

To analyze the molecular mechanism of Prunella vulgaris L. (PV) in the treatment of papillary thyroid carcinoma (PTC) by using network pharmacology combined with molecular docking verification. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database was used to predict the main active components of PV, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, PubChem, and Swiss Target Prediction databases were used to obtain the corresponding targets of all active components. Targets collected for PTC treatment through Gene Cards, Digest and Online Mendelian Inheritance in Man databases respectively. The Search Tool for the Retrieval of Interaction Gene/Protein database was used to obtain the interaction information between proteins, and the topology analysis and visualization were carried out through Cytoscape 3.7.2 software (https://cytoscape.org/). The R package cluster profiler was used for gene ontology and Kyoto encyclopedia of genes and genomes analysis. The "active ingredient-target-disease" network was constructed by using Cyto scape 3.7.2, and topological analysis was carried out to obtain the core compound. The molecular docking was processed by using Discovery Studio 2019 software, and the core target and active ingredient were verified. The inhibition rate was detected by CCK8 method. Western blot was used to detect the expression levels of kaempferol anti-PTC related pathway proteins. A total of 11 components and 83 corresponding targets in the component target network of PV, of which 6 were the core targets of PV in the treatment of PTC. It was showed that quercetin, luteolin, beta (ß)-sitosterol, kaempferol may be the core components of PV in the treatment of PTC. vascular endothelial growth factor A, tumor protein p53, transcription factor AP-1, prostaglandin endoperoxidase 2, interleukin 6, and IL-1B may be important targets for the treatment of PTC. The main biological processes mainly including response to nutrient levels, response to xenobiotic stimulus, response to extracellular stimulus, external side of plasma membrane, membrane raft, membrane microdomain, serine hydrolase activity, serine-type endopeptidase activity, antioxidant activity, etc IL-17 signaling pathway, and PI3K-Akt signaling pathway may affect the recurrence and metastasis of PTC. Kaempferol may significantly reduce the activity of Papillary cells of human thyroid carcinoma bcpap cell lines cells compared with quercetin, luteolin, ß-sitosterol. Kaempferol may reduce the protein expression levels of interleukin 6, vascular endothelial growth factor A, transcription factor AP-1, tumor protein p53, 1L-1B and prostaglandin endoperoxidase 2, respectively. PV has the characteristics of multi-components, multi-targets and multi- pathways in the treatment of PTC, which network pharmacology help to provides a theoretical basis for the screening of effective components of PV and further research.

Investigation on the Mechanisms of Zanthoxylum bungeanum for Treating Diabetes Mellitus Based on Network Pharmacology, Molecular Docking, and Experiment Verification.

Objective: The aim of the study was to explore the potential mechanism of Zanthoxylum bungeanum in the treatment of diabetes mellitus (DM) using network pharmacology. Methods: The DrugBank database and TCMSP platform were used to search for the main chemical components and their targets of Zanthoxylum bungeanum, and the genes related to diabetes mellitus were obtained from the genecards database. Import the data into the Venny 2.1.0 platform for intersection analysis to obtain the Zanthoxylum bungeanum-DM-gene dataset. The protein-protein interaction (PPI) analysis of Zanthoxylum bungeanum-DM gene was performed using the String data platform, and the visualization and network topology analysis were performed using Cytoscape 3.8.2. The KEGG pathway enrichment and biological process of GO enrichment analysis were carried out using the David platform. The active ingredients and key targets of Zanthoxylum bungeanum were molecularly docked to verify their biological activities by using Discovery Studio 2019 software. Zanthoxylum bungeanum was extracted and isolated by ethanol and dichloromethane. HepG2 cells were cultured, and cell viability assay was utilized to choose the suitable concentration of Zanthoxylum bungeanum extract (ZBE). The western blot assay was used for measuring the expression of AKT1, IL6, HSP90AA1, FOS, and JUN proteins in HepG2 cells. Results: A total of 5 main compounds, 339 targets, and 16656 disease genes were obtained and retrieved, respectively. A total of 187 common genes were screened, and 20 core genes were finally obtained after further screening. The antidiabetic active ingredients of Zanthoxylum bungeanum are kokusaginin, skimmianin, diosmetin, beta-sitosterol, and quercetin, respectively. The main targets for its antidiabetic effect are AKT1, IL6, HSP90AA1, FOS, and JUN, respectively. GO enrichment analysis revealed that the biological process of Zanthoxylum bungeanum and DM is related to a positive regulation of gene expression, positive regulation of transcription, positive regulation of transcription from RNA polymerase II promoter, response to drug, positive regulation of apoptotic process, and positive regulation of cell proliferation, etc. KEGG enrichment analysis revealed that common biological pathways mainly including the phospholipase D signaling pathway, MAPK signaling pathway, beta-alanine metabolism, estrogen signaling pathway, PPAR signaling pathway, and TNF signaling pathway. Molecular docking results showed that AKT1 with beta-sitosterol and quercetin, IL-6 with diosmetin and skimmianin, HSP90AA1 with diosmetin and quercetin, FOS with beta-sitosterol and quercetin, and JUN with beta-sitosterol and diosmetin have relatively strong binding activity, respectively. Experiment verification results showed that DM could be significantly improved by downregulating the expression of AKT1, IL6, HSP90AA1, FOS, and JUN proteins after being treated at concentrations of 20 µmol/L and 40 µmol/L of ZBE. Conclusion: The active components of Zanthoxylum bungeanum mainly including kokusaginin, skimmianin, diosmetin, beta-sitosterol, and quercetin. The therapeutic effect of Zanthoxylum bungeanum on DM may be achieved by downregulating core target genes including AKT1, IL6, HSP90AA1, FOS, and JUN, respectively. Zanthoxylum bungeanum is an effective drug in treatment of DM related to the above targets.

Effect of metformin on nonalcoholic fatty liver based on meta-analysis and network pharmacology.

BACKGROUND: Whether metformin is related to nonalcoholic fatty liver disease (NAFLD) is controversial. Our aim was to investigate the relationship between metformin and NAFLD that may predict the metformin potential of these lesions and new prevention strategies in NAFLD patients. METHODS: The meta-analysis was analyzed by Revman 5.3 softwares systematically searched for works published through July 29, 2022. Network pharmacology research based on databases, Cytoscape 3.7.1 software and R software respectively. RESULTS: The following variables were associated with metformin in NAFLD patients: decreased of alanine aminotransferase (ALT) level (mean difference [MD] = -10.84, 95% confidence interval [CI] = -21.85 to 0.16, P = .05); decreased of aspartate amino transferase (AST) level (MD = -4.82, 95% CI = -9.33 to -0.30, P = .04); decreased of triglyceride (TG) level (MD = -0.17, 95% CI = -0.26 to -0.08, P = .0002); decreased of total cholesterol (TC) level (MD = -0.29, 95% CI = -0.47 to -0.10, P = .003); decreased of insulin resistance (IR) level (MD = -0.42, 95% CI = -0.82 to -0.02, P = .04). In addition, body mass index (BMI) (MD = -0.65, 95% CI = -1.46 to 0.16, P = .12) had no association with metformin in NAFLD patients. 181 metformin targets and 868 NAFLD disease targets were interaction analyzed, 15 core targets of metformin for the treatment of NAFLD were obtained. The effect of metformin on NAFLD mainly related to cytoplasm and protein binding, NAFLD, hepatitis B, pathway in cancer, toll like receptor signaling pathway and type 2 diabetes mellitus (T2DM). The proteins of hypoxia inducible factor-1 (HIF1A), nuclear factor erythroid 2-related factor (NFE2L2), nitric oxide synthase 3 (NOS3), nuclear receptor subfamily 3 group C member 1 (NR3C1), PI3K catalytic subunit alpha (PIK3CA), and silencing information regulator 2 related enzyme 1 (SIRT1) may the core targets of metformin for the treatment of NAFLD. CONCLUSION: Metformin might be a candidate drug for the treatment of NAFLD which exhibits therapeutic effect on NAFLD patients associated with ALT, AST, TG, TC and IR while was not correlated with BMI. HIF1A, NFE2L2, NOS3, NR3C1, PIK3CA, and SIRT1 might be core targets of metformin for the treatment of NAFLD.

Effect of Probiotics Therapy on Nonalcoholic Fatty Liver Disease.

Objective: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in the world. The pathogenesis of NAFLD is complex and multifactorial. Clinical studies have shown that alterations in the gut microbiota play a key role in NAFLD. The purpose of this study was to analyze the effect of probiotic supplementation on the treatment of NAFLD patients based on various indicators. Methods: We conducted a meta-analysis investigating the relationship between NAFLD and probiotic supplementation. Embase, PubMed, and Web of Science databases were searched by computer, and then, eligible studies were identified. Finally, a total of high-quality randomized controlled trials were selected involving 1403 participants. Meta-analysis was performed using the RevMan 5.3 software which was systematically searched for works published through Dec. 1, 2021, in the present study. Results: The meta-analysis results showed that the probiotics supplementation improved hepatocyte injury and significantly reduced the level of ALT (P = 0.00001), AST (P = 0.0009), GGT (P = 0.04), TG (P = 0.01), LDL-C (P = 0.0005), HDL-C (P = 0.0002), insulin (P = 0.003), IR (P = 0.03), BMI (P = 0.03), TNF-α (P = 0.03), and CRP (P = 0.02), respectively, in NAFLD patients. Conclusion: The present study suggests that probiotics therapy may improve liver enzyme levels, regulated lipid metabolism, reduced insulin resistance, and improved inflammation in NAFLD patients. It supports the potential role of probiotics supplementation in the treatment of NAFLD.

Incaspitolide A isolated from Carpesium cernuum L. inhibits the growth of prostate cancer cells and induces apoptosis via regulation of the PI3K/Akt/xIAP pathway.

Carpesium cernuum L. is a traditional medicine primarily used in Southwestern China, and it has been shown to exhibit a range of biological properties, including anti-inflammatory and antitumor activities. Incaspitolide A (IA) is a sesquiterpene isolated from C. cernuum L. The aim of the present study was to investigate the antiproliferative effects of IA on PC-3 prostate cancer cells and determine the underlying mechanism. Results from a Cell Counting Kit-8 assay demonstrated that IA significantly reduced the numbers of viable PC-3 cells in a time and dose-dependent manner. Phase-contrast microscopy revealed that the number and morphology of cells were markedly altered. Hoechst and EdU staining assays showed that IA reduced the proliferation of PC-3 cells. Flow cytometry analysis revealed that IA arrested cell cycle progression at the S phase and promoted cell apoptosis in a dose-dependent manner. Western blot analysis demonstrated that treatment with IA resulted in downregulation of phosphorylated (p-) PI3K, p-Akt, X-linked inhibitor of apoptosis (xIAP), CKD2, cyclin A2 and pro-Caspase-3 protein expression, and upregulation of cleaved poly(ADP-ribose) polymerase and P53 expression. The present results suggested that IA inhibited the growth of PC-3 cells and induced apoptosis. The underlying mechanism appeared to involve the inhibition of the PI3K/Akt/xIAP pathway. The present study indicated that IA may serve as a therapeutic for the management of prostate cancer and provided a theoretical basis for the pathogenesis of prostate cancer.

Antiprostate Cancer Activity of Ineupatolide Isolated from Carpesium cernuum L.

OBJECTIVE: The aim of the study was to investigate the antiprostate cancer effects and mechanism of ineupatolide (T-21), a natural product isolated from the Compositae plant Carpesium cernuum L., on PC-3 human prostate cancer cells. METHODS: The effect of T-21 on the proliferation of PC-3 cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell migration, and invasion experiments; the morphology of cell apoptosis was observed by Hoechst-propidium iodide staining; the effects of T-21 on PC-3 cell apoptosis and the cell cycle were evaluated by flow cytometry; and the effect of T-21 on the expression levels of phosphorylated protein kinase B (p-AKT), AKT, X-linked inhibitor of apoptosis protein (xlAP), procaspase-3, and poly (ADP-ribose) polymerase (PARP) in PC-3 cells was measured by western blotting. RESULTS: T-21 significantly inhibited the proliferation of cells, and its half-maximal inhibitory concentrations at 12, 24, and 48 h were 38.46 ± 1.01, 24.63 ± 0.70, and 7.36 ± 0.58 µM, respectively. T-21 may promote cell apoptosis in a concentration-dependent manner and block the cell cycle in the G2 and S phases. In addition, T-21 significantly reduced the protein expression levels of p-AKT, AKT, xlAP, procaspase-3, and PARP. CONCLUSION: T-21 exhibits antiproliferation effects on PC-3 cells by promoting apoptosis and arresting the cell cycle in the G2 and S phases. The possible mechanism underlying its potential therapeutic effects against prostate cancer is related to the AKT/xlAP pathway.

Costunolide isolated from Vladimiria souliei inhibits the proliferation and induces the apoptosis of HepG2 cells.

Costunolide (cos) is one of the major sesquiterpenes isolated from the ethyl acetate soluble fraction of the roots of Vladimiria souliei. In order to explore the effects and molecular mechanism of cos, the anti­proliferative and apoptotic effects of cos against the human hepatoblastoma HepG2 cell line was examined in vitro in the current study. Cell viability was measured using an MTT assay, and IC50 values (indicating the concentration required to achieve half­maximal inhibition) were calculated to detect the inhibitory effect of cos on HepG2 cell growth. Cell morphology was subsequently observed under an inverted microscope, and cell cycle distribution and apoptosis were detected using flow cytometric analysis. In addition, changes in the protein expression levels of B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein (Bax), and caspases­3, ­8 and ­9 were detected by western blotting. The results of cell analyses indicated that cos treatment inhibited the proliferation and promoted the apoptosis of HepG2 cells in vitro. Cos markedly induced HepG2 cell apoptosis by arresting the cell cycle at the G2/M phase in a dose­dependent manner. In terms of the underlying mechanism, cos was revealed to inhibit the anti­apoptotic capacity of the cells, possibly via upregulating the expression levels of Bax protein and caspases­3, ­8 and ­9, and downregulating the expression of Bcl­2 protein. Taken together, the results of the present study indicate that cos may be a promising candidate for liver cancer therapy, and have provided an insight into the mechanism of action involved in its anti­cancer properties.

Protective Effects of Costunolide Against D-Galactosamine and Lipopolysaccharide-Induced Acute Liver Injury in Mice.

Costunolide, a sesquiterpene isolated from Vladimiria souliei (Franch.) Ling, is known to exhibit anti-inflammatory, anti-viral, and anti-tumor activities. However, the effects of costunolide on liver injury are poorly understood. The current study aimed to investigate the hepatoprotective effects of costunolide against lipopolysaccharide (LPS) and D-galactosamine-induced acute liver injury (ALI) in mice. The results indicated that costunolide (40 mg/kg) could significantly improve the pathological changes of hepatic tissue, and reduced the LPS and D-galactosamine-induced increases of alanine aminotransferase (from 887.24 ± 21.72 to 121.67 ± 6.56 IU/L) and aspartate aminotransferase (from 891.01 ± 45.24 to 199.94 ± 11.53 IU/L) activities in serum. Further research indicated that costunolide significantly reduced malondialdehyde content (from 24.56 ± 1.39 to 9.17 ± 0.25 nmol/ml) and reactive oxygen species (from 203.34 ± 7.68 to 144.23 ± 7.12%), increased the activity of anti-oxidant enzymes superoxide dismutase (from 153.74 ± 10.33 to 262.27 ± 8.39 U/ml), catalase (from 6.12 ± 0.30 to 12.44 ± 0.57 U/ml), and total anti-oxidant capacity (from 0.64 ± 0.06 to 6.29 ± 0.11 U/ml) in hepatic tissues. Western blot results revealed that costunolide may trigger the anti-oxidative defense system by inhibiting kelch-like ECH-associated protein 1 and nuclear factor-related factor 2 (cytosol), increasing nuclear factor-related factor 2 (nucleus), heme oxygenase-1 and NAD (P) H quinone oxidoreductase 1 activity. Moreover, costunolide significantly decreased the protein expression of proinflammatory cytokines including interleukin 1ß, interleukin 6, and tumor necrosis factor. Pretreatment with costunolide could reduce the expression of toll-like receptor 4, myeloid differentiation factor 88, p65 (Nucleus), phosphorylated IκB kinase α/ß, inhibitor of nuclear factor kappa-B kinase, inhibitor kappa Bα and prevent the expression of phosphorylated inhibitor kappa B kinase which repressed translocation of p65 from cytoplasm to nucleus. In addition, pretreatment with costunolide also inhibited hepatocyte apoptosis by reducing the expression of B-cell lymphoma 2 associated X, cytochrome C, cysteinyl aspartate specific proteinase 3, cysteinyl aspartate specific proteinase 8 and cysteinyl aspartate specific proteinase 9, and by increasing B-cell lymphoma 2. From the above analysis, the protective effects of costunolide against LPS and D-galactosamine-induced ALI in mice may be attributed to its anti-oxidative activity in nuclear factor-related factor 2 signaling pathways, anti-inflammatory suppression in nuclear factor-kappa B signaling pathways, and inhibition of hepatocyte apoptosis. Thus, costunolide may be a potential therapeutic agent in attenuating LPS and D-galactosamine -induced ALI in the future.

Profiling of anthocyanins in transgenic purple-fleshed sweet potatoes by HPLC-MS/MS.

BACKGROUND: Anthocyanins in purple-fleshed sweet potato (PSP) are beneficial to human health. The leaf color (Lc) gene is a transcription factor involved in regulating anthocyanin biosynthesis. The anthocyanin profiles of wild-type PSP of Ayamurasaki and its three Lc-transgenic lines were investigated by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). In vitro antioxidant activities of wild-type and Lc-transgenic lines, including reducing power activity, DPPH radical scavenging activity, hydroxyl radical scavenging activity, linoleic acid autoxidation inhibition activity, ABTS free radical scavenging activity and oxygen radical absorbance capacity activity, were measured. RESULTS: The results showed that the total anthocyanin contents increased 1.5-1.9 times in three transgenic lines compared with that in wild-type PSP. Seventeen anthocyanins were found in wild-type PSP, while 19 in Lc-transgenic lines including cyanidin-based, peonidin-based and pelargonidin-based anthocyanins. Three pelargonidin-based anthocyanins were detected in three Lc-transgenic lines. Among them, the relative contents of cyanidin-based and pelargonidin-based anthocyanins increased 1.9-2.0 and 3.4-4.5 times respectively, while peonidin-based anthocyanins decreased 1.8-1.9 times in Lc-transgenic lines, compared with wild-type PSP. PSP from wild-type Ayamurasaki and three Lc-transgenic lines exhibited potent antioxidant activities, whereas there was no distinct difference among them. CONCLUSION: The transgene Lc significantly increased the content of total anthocyanins and remarkably changed the anthocyanin profiles in Ayamurasaki. Such novel and high content of anthocyanins obtained in the Lc-transgenic lines with potent antioxidant activities may provide unique functional products with potential helpful for human health. © 2017 Society of Chemical Industry.

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.