Icariin inhibits hypertrophy by regulation of GPER1 and CaMKII/HDAC4/MEF2C signaling crosstalk in ovariectomized mice.

Icariin (ICA), a flavonoid phytoestrogen, was isolated from traditional Chinese medicine Yin Yang Huo (Epimedium brevicornu Maxim.). Previous studies reporting the cardioprotective effects of ICA are available; however, little is known about the impact of ICA on cardioprotection under conditions of reduced estrogen levels. This study aimed to provide detailed information regarding the antihypertrophic effects of ICA in ovariectomized female mice. Female mice were subjected to ovariectomy (OVX) and transverse aortic constriction and then orally treated with ICA at doses of 30, 60 or 120 mg/kg/day for 4 weeks. Morphological assessments, echocardiographic parameters, histological analyses, and immunofluorescence were performed to evaluate cardiac hypertrophy. Cardiomyocytes from mice or rats were stimulated using phenylephrine, and cell surface and hypertrophy markers were tested using immunofluorescence and qPCR. Western blotting, qPCR, and luciferase reporter gene assays were used to assess the expression of proteins and mRNA and further investigate the proteins related to the G-protein coupled estrogen receptor (GPER1) and CaMKII/HDAC4/MEF2C signaling pathways in vivo and in vitro. ICA blocks cardiac hypertrophy induced by pressure overload in OVX mice. Additionally, we demonstrated that ICA activated GPER1 and inhibited the nuclear export or promoted the nuclear import of histone deacetylase 4 (HDAC4) through regulation of phosphorylation of calmodulin-dependent protein kinase II (CaMKII) and further improved the repression of myocyte enhancer factor-2C (MEF2C). ICA ameliorated cardiac hypertrophy in OVX mice by activating GPER1 and inhibiting the CaMKII/HDAC4/MEF2 signaling pathway.

Stachydrine hydrochloride ameliorates cardiac hypertrophy through CaMKII/HDAC4/MEF2C signal pathway.

Stachydrine hydrochloride (Sta), an activated alkaloid, is isolated from traditional Chinese medicine Yimucao. In previous studies, the cardioprotective effects of Sta were found in our laboratory. However, the underling mechanisms of Sta is not fully elucidated. The aim of this study was to provide a detailed account of the anti-hypertrophic effects of Sta on transcriptional regulation. In vivo, C57BL/6J mice were subjected to transverse aortic constriction (TAC) and were orally treated with Sta. Morphological assessments, echocardiographic parameters, histological analyses and immunofluorescence were used to evaluate cardiac hypertrophy. In vitro, cardiomyocytes were stimulated by phenylephrine (PE), and cell surface and hypertrophy markers were tested by immunofluorescence and real-time polymerase chain reaction (RT-PCR). Moreover, western blotting, RT-PCR and luciferase reporter genes were used to assess the expression of proteins, mRNA and the activity of the CaMKII/HDAC4/MEF2C signal pathway in vivo and in vitro. We found that Sta blocked cardiac hypertrophy induced by pressure overload. We also demonstrated that Sta inhibited nuclear export or promoted nuclear import of HDAC4 through regulation of p-CaMKII, and it further improved the repression of MEF2C. Taken together, our findings demonstrated that Sta ameliorates cardiac hypertrophy through CaMKII/HDAC4/MEF2C signal pathway.

Capsaicin ameliorates renal fibrosis by inhibiting TGF-ß1-Smad2/3 signaling.

BACKGROUND AND PURPOSE: Chronic kidney disease (CKD), characterized by renal fibrosis, is a global refractory disease with few effective therapeutic strategies. It has been reported that capsaicin exerts many pharmacological effects including liver and cardiac fibrosis. However, whether capsaicin plays a therapeutic role in renal fibrosis remains unclear. METHODS: We investigated antifibrotic effects of capsaicin in two mouse renal fibrosis models as follows: C57BL/6J mice were subjected to unilateral ureteral obstruction (UUO) and fed with an adenine-rich diet. We uncovered and verified the mechanisms of capsaicin in human proximal tubular epithelial cells (HK2). We mainly used histochemistry, immunohistochemistry and immunofluorescence staining, western blot assay, biochemical examination and other tools to examine the effects of capsaicin on renal fibrosis and the underlying mechanisms. RESULTS: Capsaicin treatment significantly alleviated fibronectin and collagen depositions in the tubulointerstitium of the injured kidneys from UUO and adenine-fed mice. Meanwhile, capsaicin treatment obviously reduced α-SMA expression. Moreover, capsaicin treatment dramatically protected against the phenotypic alteration of tubular epithelial cells by increasing E-cadherin expression and decreasing vimentin expression during renal fibrosis. Mechanistically, capsaicin treatment effectively suppressed α-SMA and vimentin expressions but promoted E-cadherin expression in HK2 cells mainly through the inhibition of TGF-ß1-Smad2/3 signaling. CONCLUSION: Capsaicin significantly ameliorated renal fibrosis possibly by retarding the activation of myofibroblasts and protecting against the phenotypic alteration of tubular epithelial cells mainly through the inhibition of TGF-ß1-Smad2/3 signaling. Thus, our findings may provide a new insight into the clinical application of capsaicin in renal fibrosis.

Multi-nutrient stoichiometry of Chinese hickory (Carya cathayensis) saplings: plant organs vary in their response to nitrogen fertilization.

Nitrogen (N) enrichment from excessive fertilization in managed forests affects biogeochemical cycles on multiple scales, but our knowledge of how N availability shifts multi-nutrient stoichiometries (including macronutrients: N, phosphorus, potassium, calcium, magnesium and micronutrients: manganese, iron and zinc) within and among organs (root, stem and leaf) remains limited. To understand the difference among organs in terms of multi-nutrient stoichiometric homeostasis responding to N fertilization, a six-level N supply experiment was conducted through a hydroponic system to examine stem growth, multi-nutrient concentrations and stoichiometric ratios in roots, stems and leaves of 2-year-old Chinese hickory (Carya cathayensis Sarg.) saplings. Results showed that N supply significantly enhanced leaf length, width, basal diameter and sapling height. Increasing the rates of N also significantly altered multi-nutrient concentrations in roots, stems and leaves. Macronutrients generally respond more positively than micronutrients within organs. Among organs, leaves and stems generally responded more actively to N supply than roots. The stoichiometric ratios of nutrients within different organs changed significantly with N supply, but their direction and degree of change varied by organ. Specifically, increased N supply reduced the ratios of both macronutrients and micronutrients to N in plant organs, while increased N supply elevated the ratios of P to other nutrients. With N fertilization, ratios of micronutrients decreased in leaves and stems and increased in roots. In particular, leaf N and stem Mn stoichiometries responded strongly to N availability, indicating stimulated N uptake but a decreased risk of Mn2+ accumulation to excessive N. Overall, Chinese hickory saplings responded positively to increasing N availability in terms of stem growth, but the multi-nutrient stoichiometric homeostasis was distinctively organ-dependent. These results are expected to enhance our understanding of N-induced changes in homeostasis of multiple nutrients at the organ level and may offer new insights into how plants adapt to increasing N fertilization.

Senkyunolide H protects PC12 cells from OGD/R-induced injury via cAMP-PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Senkyunolide H (SNH) is a bioactive phthalide isolated from Ligusticum chuanxiong Hort rhizome and was reported to have multiple pharmacological effects. AIM OF THE STUDY: The study was performed to verify the potency of SNH protecting PC12 cells from oxygen glucose deprivation/reperfusion (OGD/R)-induced injury and to elucidate the underlying mechanisms. MATERIALS AND METHODS: OGD/R model was established in PC12 cells and the cell viability was measured by MTT assay. The cell morphology was observed using scanning electron microscope (SEM). The potential targets of SNH and related targets of OGD/R were screened, and a merged protein-protein interaction (PPI) network of SNH and OGD/R was constructed based on the network pharmacology analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway analysis. Intracellular cAMP level and the protein expression levels were measured to elucidate the underlying mechanisms. RESULTS: SNH pretreatment protected PC12 cells against OGD/R-induced cell death. SNH also significantly protected the cell protrusion. A merged PPI network was constructed and the shared candidate targets significantly enriched in cAMP signaling pathway. The level of intracellular cAMP and the protein level of p-CREB, p-AKT, p-PDK1 and PKA protein were up-regulated after the treatment of SNH compared with OGD/R modeling. CONCLUSIONS: The present study indicated that SNH protected PC12 cells from OGD/R-induced injury via cAMP-PI3K/AKT signaling pathway.

Clinical characteristics and viral shedding kinetics of 38 asymptomatic patients with coronavirus disease 2019: A retrospective observational study.

ABSTRACT: This study aims to investigate the clinical characteristics and viral shedding kinetics of asymptomatic patients with coronavirus disease 2019 (COVID-19).The data of 38 asymptomatic patients positive for SARS-CoV-2 nucleic acid were collected from February to March 2020 in Tuanfeng County, Huanggang, Hubei, China. The epidemiology, laboratory examination, chest imaging, viral nucleic acid test results, clinical characteristics, and viral shedding time were summarized in this retrospective study.The study included 20 family members of patients with COVID-19, 10 medical personnel participating in COVID-19 treatment or working in a fever clinic, 6 personnel from quarantine places, 1 individual with a close contact history with confirmed patients, and 1 local epidemic prevention personnel. All were positive for SARS-CoV-2 nucleic acid. The white blood cell (WBC) count, the absolute value of lymphocytes, C-reactive protein (CRP), and D-dimer were normal. Pneumonia manifestations were not found in the chest computed tomography (CT) scan of 36 patients; the remaining 2 cases included a 1-year-old child and a pregnant woman, and they did not undergo chest CT. The viral shedding time was 6 days.All asymptomatic patients with COVID-19 had a history of close contact or exposure. Laboratory tests were normal. Chest imaging did not show any pneumonia manifestation. The viral shedding time was <10 days, which is shorter than that of patients with COVID-19. A timely discovery of such asymptomatic infections is crucial for blocking the spread of the virus and strengthening the prevention and control measures.

Xinjiang herbal tea exerts immunomodulatory activity via TLR2/4-mediated MAPK signaling pathways in RAW264.7 cells and prevents cyclophosphamide-induced immunosuppression in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: A multi-herb Chinese medicinal formula consisting of a variety of medicinal and edible materials has long been consumed as a hot drink and immune enhancer for its efficiency to increase disease resistance in Xinjiang, China. However, no fundamental data has been collected associated with traditional consumption. The present work was designed to evaluate the immunostimulatory role of Xinjiang herbal tea (XMT-WE) in RAW 264.7 macrophages and cyclophosphamide (CTX)-induced immunosuppression mice model. MATERIALS AND METHODS: RAW 264.7 cells were treated with various concentrations of XMT-WE. Nitric oxide (NO) levels were determined using Griess reagents, and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α were investigated with a cytometric bead array kit. The effects on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and TNF-α were investigated. Furthermore, activation of nuclear factor (NF)-κB and AP-1 mitogen-activated protein kinase (MAPK) signaling pathways was investigated. RESULTS: Pre-treatment with XMT-WE significantly increased secretion of NO, IL-6, and TNF-α. In addition, XMT-WE markedly increased expression of iNOS, COX-2, and TNF-α as well as AP-1 and NF-κB translocation from the cytoplasm into the nucleus, which was associated with an increase of phosphorylated ERK, JNK, and p38 as well as membrane receptors such as toll-like receptor (TLR) 2 and TLR4. Moreover, XMT-WE promoted the secretion of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in cyclophosphamide (CTX)-induced immunosuppressive mice. CONCLUSION: These results indicated that XMT-WE at 50 µg/ml exerts immunomodulatory activity via TLR2/4-mediated MAPK signaling pathways in RAW 264.7 cells. Furthermore, in vivo experiments revealed that XMT-WE at the dose of 50 and 100 mg/kg strongly stimulated inflammatory cytokines.

Extraction of Phenylethanoid Glycosides from Cistanche tubulosa by High-Speed Shearing Homogenization Extraction.

Background: Cistanche tubulosa is a well-known traditional Chinese medicine originating in Xinjiang. It is widely distributed in northern Africa, India, etc. Objective: The major bioactive component of C. tubulosa is phenylethanoid glycosides (PhGs). Echinacoside and acteoside are the indicative components for the determination of PhGs and are mainly used for liver protection, immune protection, etc. Therefore, it is very important to extract the PhGs from C. tubulosa. Methods: In this study, the ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and high-speed shearing homogenization extraction (HSHE) methods were compared. Furthermore, the extraction conditions of the HSHE method were optimized. Results: The results showed that the HSHE method was better than both the UAE and MAE methods, and the optimal extraction parameters of HSHE were an ethanol concentration of 50%, an extraction temperature of 70°C, a rotation speed of 16 000 rpm, an extraction time of 2 min, a solid-to-liquid ratio of 1:9, and one extraction cycle. The yields of echinacoside and acteoside were 1.366 and 0.519%, respectively, and the transfer rates of echinacoside and acteoside reached 87 and 94%, respectively. Conclusions: It can be concluded that the HSHE method is a simple, rapid, and efficient technique for extracting PhGs from C. tubulosa. Highlights: An efficient and ecofriendly HSHE method has been investigated for the extraction of PhGs from C. tubulosa. The optimum conditions of the HSHE method for the extraction of PhGs from C. tubulosa were obtained. This research provides a new method for the industrial extraction of PhGs from C. tubulosa.

Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells.

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1ß at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.

[Inhibition of Combination of Icaritin and Doxorubicin on Human Osteosarcoma MG-63 Cells in vitro].

OBJECTIVE: To explore the inhibition and molecular mechanism of icaritin (ICT) combined doxorubicin (DOX) on human osteosarcoma MG-63 cells in vitro. METHODS: The control group, ICT groups (10, 20, 40, 80, and 160 µmol/L), DOX groups (1, 2, 4, 8, and 16 µg/mL), and combination groups (20 µmol/ L ICT +1 µg/mL DOX, 20 µmol/L ICT +2 µg/mL DOX, 20 µmol/L ICT +4 µg/mL DOX, 40 µmol/L ICT +1 µg/mL DOX, 40 µmol/L ICT +2 µg/mL DOX, 40 µmol/L ICT +4 µg/mL DOX, 80 µmol/L ICT +1 µg/mL DOX, 80 µmol/L ICT +2 µg/mL DOX, 80 µmol/L ICT +4 µg/mL DOX) were set up. Human osteosarcoma MG-63 cells were respectively cultured and their effects on morphological changes were observed using inverted phase contrast microscope after 24-and 48-h intervention. The cell proliferation inhibition rate of each group was de- termined using CCK-8, and IC50 calculated. The MG-63 apoptosis rate was detected using Annexin V-FITC/ PI double dye flow cytometry. Expression levels of bcl-2, caspase-3, and p21 were detected using RT-PCR. RESULTS: ICT and DOX could obviously inhibit the proliferation of MG-63 cell. Along with ICT concentration increasing from 10 µmol/L to 160 µmol/L, the cell proliferation inhibition rate also increased gradually from 9.67% ± 3.62% to 89.18% ± 9.66%. The IC50 was 46.93 µmol/L and 3.87 µg/mL respectively. ICT and DOX could cause either early or late stage apoptosis, down-regulate Bcl-2 gene expression, and up-regulate gene expressions of Caspase-3 and p21 respectively (P < 0.05). Aforesaid changes were more obviously seen in combination groups than in lCT groups and DOX groups (P < 0.05). CONCLUSION: CT combined DOX had additive or synergistic inhibition effect for the proliferation of osteosarcoma MG-63 cells, which might be related with regulating gene expressions of bcl-2, caspase-3, and p21.

Gene cloning, sequence analysis, and expression profiles of a novel ß-ring carotenoid hydroxylase gene from the photoheterotrophic green alga Chlorella kessleri.

In this study, a full-length complementary DNA (cDNA) sequence of ß-ring carotenoid hydroxylase (CHY), designated Ckecyp97a1, was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends (RACE) methods. The cloned Ckecyp97a1 cDNA was 2,264-bp in length, and contained an open reading frame (ORF) of 1,944-bp with 5'-terminal untranslated region (UTR) of 66-bp and 3'-terminal UTR of 254-bp and encoded a ß-ring CHY protein of 647 amino acids. The deduced protein had a calculated molecular mass of 71.43 kDa with an estimated isoelectric point (pI) of 6.72. Multiple sequence alignment and phylogenetic analysis revealed that Ckecyp97a1 was homologs to known chloroplastic cytochrome P450 (P450) CHY. The typical catalytic motifs of the P450 were highly conserved in the protein sequences of CkeCYP97A1. The Ckecyp97a1 transcriptional expression and carotenoids accumulation were observed under high light (HL) of different wavelengths (white: 390-770 nm and blue: 420-500 nm). The results revealed that Ckecyp97a1 transcript increased strongly throughout the course of the HL illumination treatment (22-70 h) under white HL treatment, while decreased during 10-58 h under blue HL treatment. The concentrations of lutein, α-carotene, and ß-carotene were relatively steady and below the control level under both treatments. The zeaxanthin concentration was higher under white HL treatment than those under control and blue HL treatments. Ckecyp97a1 gene showed different expression patterns under different light wavelengths treatments. The data obtained in this study demonstrates that CkeCYP97A1 is the enzyme responsible for carotenoid hydroxylation involved in HL acclimation for photoheterotrophic green alga Chlorella kessleri CGMCC 4917.