Gentiopicroside ameliorates the lipopolysaccharide-induced inflammatory response and hypertrophy in chondrocytes.

PURPOSE: This study aimed to evaluate the protective effects of gentiopicroside against lipopolysaccharide-induced chondrocyte inflammation. METHODS: SW 1353 chondrosarcoma cells were stimulated with LPS (5 µg/ml) for 24 h and treated with different concentrations of gentiopicroside (GPS) for 24 h. The toxic effects of GPS on chondrocytes were determined using a CCK-8 assay and EdU staining. Western blotting, qPCR, and immunofluorescence analysis were used to examine the protective effect of GPS against the inflammatory response in chondrocytes induced by lipopolysaccharide (LPS). One-way ANOVA was used to compare the differences between the groups (significance level of 0.05). RESULTS: The CCK-8 results showed that 10, 20 and 40 µM GPS had no significant toxic effects on chondrocytes; GPS effectively reduced the production of IL-1ß and PGE2, reversed LPS-induced extracellular matrix degradation in cartilage by inhibiting the Stat3/Runx2 signaling pathway, and suppressed the hypertrophic transformation of SW 1353 chondrosarcoma cells. CONCLUSION: Our study demonstrated that GPS significantly inhibited the LPS-induced inflammatory response and hypertrophic cellular degeneration in SW 1353 chondrosarcoma cells and is a valuable traditional Chinese medicine for the treatment of knee osteoarthritis.

Linderane Attenuates Complete Freund's Adjuvant-Induced Pain and Anxiety in Mice by Restoring Anterior Cingulate Cortex Microglia M2 Polarization through Activating Cannabinoid 2 Receptor.

Chronic pain is a common condition that causes negative emotions as the disease progresses. The anterior cingulate cortex (ACC) is a key region in the integration of nociceptive perception and emotional response in chronic pain. Linderane (LDR) is an active ingredient from Linderae radix, a traditional Chinese medicine with anti-inflammatory, analgesic, and antibacterial properties. In this study, the analgesic and antianxiety effects of LDR were evaluated using a complete Freund's adjuvant (CFA)-induced inflammatory pain model in C57BL/6 male mice. Mechanical and thermal pain sensitivity were measured through plantar mechanical analgesia and hot plate apparatus, and anxiety-like behavior was evaluated by open field and elevated plus maze tests. The results showed that LDR-alleviated CFA-induced pain and anxiety, reduced the release of inflammatory cytokines, and inhibited ACC microglial activation. Target prediction, molecular docking, and cellular thermal shift assay demonstrated that LDR could bind to the cannabinoid 2 receptor (CB2R), a key component of the endocannabinoid system with an important role in regulating pain and related emotions. Moreover, both the analgesic effect of LDR and its regulation of microglia polarization were reversed by a CB2R antagonist (SR144528) treatment. Therefore, our results suggested that LDR exerted analgesic effects by regulating microglial polarization in ACC via CB2R activation.

Praeruptorin C alleviates cognitive impairment in type 2 diabetic mice through restoring PI3K/AKT/GSK3ß pathway.

Diabetic encephalopathy is a common consequence of diabetes mellitus that causes cognitive dysfunction and neuropsychiatric disorders. Praeruptorin C (Pra-C) from the traditional Chinese medicinal herb Peucedanum praeruptorum Dunn. is a potential antioxidant and neuroprotective agent. This study was conducted to investigate the molecular mechanisms underlying the effect of Pra-C on diabetic cognitive impairment. A novel object recognition test and the Morris water maze test were performed to assess the behavioral performance of mice. Electrophysiological recordings were made to monitor synaptic plasticity in the hippocampus. A protein-protein interaction network of putative Pra-C targets was constructed, and molecular docking simulations were performed to predict the potential mechanisms of the action of Pra-C. Protein expression levels were detected by western blotting. Pra-C administration significantly lowered body weight and fasting blood glucose levels and alleviated learning and memory deficits in type 2 diabetic mice. Network pharmacology and molecular docking results suggested that Pra-C affects the PI3K/AKT/GSK3ß signaling pathway. Western blot analysis confirmed significant increases in phosphorylated PI3K, AKT, and GSK3ß levels in vivo and in vitro upon Pra-C administration. Pra-C alleviated cognitive impairment in type 2 diabetic mice by activating PI3K/AKT/GSK3ß pathway.

Non-Invasive Thermal Therapy for Tissue Engineering and Regenerative Medicine.

Owing to the development of nanotechnology and noninvasive treatment, thermal therapy in combination with external stimuli has been applied for tissue engineering and regenerative medicine (TERM), which has attracted more and more attention in recent years. In this review, the recent progress of applying a variety of non-invasive thermal therapeutic modalities for TERM, including photothermal therapy, magnetic thermotherapy, and ultrasound thermotherapy, as well as other thermal therapeutics are discussed. The parameters and conditions that need to be considered and regulated to realize a well-controlled thermal therapy for tissue regeneration are also discussed. Afterwards, the current concerns and challenges of putting thermal therapy into clinical applications are pointed out. At last, perspectives are provided for the future development directions, aiming to providing opportunities and a novel pathway for TERM.

Rubinoboletus ballouii polysaccharides exhibited immunostimulatory activities through toll-like receptor-4 via NF-κB pathway.

The biological activities of water-soluble components of edible mushroom Rubinoboletus ballouii (RB) were seldom reported. Polysaccharides of RB (RBP) were prepared and well-characterized using chemical analyses. The immunomodulatory properties of RBP were investigated using human monocyte-derived dendritic cells (moDC) in vitro, and cyclophosphamide (CTX)-induced immunosuppressive mouse model. Results showed that RBP was found to contain 80.6% (w/w) of neutral sugars including D-fucose, D-mannose, D-glucose and D-galactose (1.7:1.4:1.0:1.8), and 12.5% (w/w) of proteins, which composed of glutamine, threonine, serine, etc. RBP could promote the maturation of moDC and increase the secretion of IL-12p40, IL-10, and TNF-α. Furthermore, the stimulation of IL-12p40 production was inhibited by pretreatment with toll-like receptor (TLR)-4 blocker or NF-κB pathway blocker, suggesting that the activation of moDC by RBP was mediated through NF-κB pathway via TLR-4 receptor. On the other hand, in CTX-treated mice, RBP restored the loss of CD34bright CD45dim hematopoietic stem cells and increased IL-2 production in sera and splenocytes culture supernatant, as well as up-regulated the percentage of CD4+ T helper lymphocyte in mice splenocytes. These findings strongly suggested that RBP are the active ingredients of RB responsible for its immunostimulatory actions and deserved to be further investigated as cancer supplements.

A Network Pharmacology Approach to Reveal the Underlying Mechanisms of Paeonia lactiflora Pall. On the Treatment of Alzheimer's Disease.

OBJECTIVE: To investigate the potential active compounds and underlying mechanisms of Paeonia lactiflora Pall. (PLP) on the treatment of Alzheimer's disease (AD) based on network pharmacology. METHODS: The active components of PLP were collected from Traditional Chinese Medicine System Pharmacology (TCMSP) database, and their possible target proteins were predicted using TCMSP, SwissTargetPrediction, and STITCH databases. The putative AD-related target proteins were identified from Therapeutic Target Database (TTD), GeneCards, and MalaCards database. The compound-target-disease network interactions were established to obtain the key targets about PLP acting on AD by network topology analysis. Then, the function annotation and signaling pathways of key targets were performed by GO and KEGG enrichment analysis using DAVID tools. Finally, the binding capacity between active ingredients and key targets was validated by molecular docking using SystemsDock tools. RESULTS: There were 7 active compounds involving in 151 predicted targets identified in PLP. Besides, a total of 160 AD-related targets were identified. Among these targets, 30 shared targets of PLP and AD were acquired. After topological analysis of the PLP potential target-AD target network, 33 key targets that were highly responsible for the therapeutic effects of PLP on AD were obtained. Further GO and KEGG enrichment analysis showed that these key targets were significantly involved in multiple biological processes and pathways which participated in cell apoptosis and inflammatory response and maintained the function of neurons to accomplish the anti-AD activity. The molecular docking analysis verified that the 7 active compounds had definite affinity with the key targets. CONCLUSIONS: The ameliorative effects of PLP on AD were predicted to be associated with regulating neural cell apoptosis, inflammatory response, and neurotrophy via various pathways such as PI3K-Akt signaling pathway, MAPK signaling pathway, and neurotrophin signaling pathway.

A combined molecular biology and network pharmacology approach to investigate the multi-target mechanisms of Chaihu Shugan San on Alzheimer's disease.

Chaihu Shugan San (CSS) is a well-known herbal formula used to nourish liver and blood, promote blood circulation and Qi flow in Traditional Chinese Medicine. Modern pharmacological studies and clinical uses showed that CSS could ameliorate cognitive dysfunction of Alzheimer's disease (AD). The present study aimed to elucidate the multi-target mechanisms of CSS on AD using network pharmacology analysis and verify its effect by biological experiments. Firstly, a total of 152 active compounds in CSS, 520 predicted biological targets and 160 AD-related targets were identified. Subsequently, the networks including herb-compound-target network, AD-target network, and CSS potential target-AD target network were constructed. 60 key targets highly responsible for the beneficial effect of CSS on AD were identified by central network topological analysis. They were significantly characterized as nuclear or cytoplasmic proteins with molecular function of protein binding. They were also enriched in various biological processes through PI3K-Akt signaling pathway, MAPK signaling pathway and HIF signaling pathway by GO function and KEGG pathway enrichment analysis. Pretreatment with CSS ameliorated Aß-induced neural cell death and reduced the number of apoptotic cells in differentiated PC12 cells. Moreover, increased phosphorylation of Akt accompanied with decreased Bax expression was found after CSS pretreatment, suggesting that Akt signaling pathway was involved in the protective effect of CSS against neural cells death. The present study systematically revealed the multi-target mechanisms of CSS on AD using network pharmacology approach, as well as validated the protective effect of CSS against Aß-induced neural cells death through Akt signaling pathway. It provided indications for further mechanistic studies and also for the development of CSS as a potential treatment for AD patients.

Excess Folic Acid Supplementation Before and During Pregnancy and Lactation Activates Fos Gene Expression and Alters Behaviors in Male Mouse Offspring.

Periconceptional folic acid (FA) supplementation is recommended to prevent neural tube defects and other birth defects. After 20 years mandate food fortification with FA, serum concentration of folate and unmetabolized FA increased significantly in the North American population. But whether excess FA intake impairs neurodevelopment and behavior is still controversial. Here, we treated mice with approximately 2.5-fold (moderate dose of FA, MFA) or 10-fold (high dose of FA, HFA) the dietary requirement of FA 1 week before mating and throughout pregnancy and lactation, and examined behaviors in adult male offspring using open field test, three-chamber sociability and social novelty test, elevated plus maze, rotarod and Morris water maze. We found that early life MFA supplementation increased long-term body weight gain in adults, elevated anxiety-like behavior, and impaired social preference, motor learning and spatial learning ability without modifying motor ability and spatial memory. In contrast, HFA supplementation only induced mild behavioral abnormality. RNA sequencing revealed that FA supplementation altered the expression of brain genes at weaning, among which Fos and related genes were significantly up-regulated in MFA mice compared with control and HFA mice. Quantitative real time-PCR (qRT-PCR) and western blots confirmed the increase of these genes. Our results suggested that FA supplementation during early life stage affected gene expression in weaning mice, and exhibited long-term impairments in adult behaviors in a dose-sensitive manner.

[Protective effect of ginsenoside Rb1 on doxorubicin-induced myocardial autophagy].

Ginsenoside Rb1 (Rb1), which is one of the main ingredients derived from Panax ginseng, has been found to have extensive pharmacological activities including antioxidant, anti-inflammatory, anticancer properties. In this study, the effect of Rb1 on doxorubicin-induced myocardial autophagy was studied with H9c2 as the study object. CCK-8 method, transmission electron microscope observation, fluorescence staining observation and Western blot were used to detect changes in H9c2 cell proliferation and autophagy after treatment. According to the results, doxorubicin could cause cell viability decrease, significant increase in the LC3-Ⅱ/LC3-I ratio and down-regulation of the expression of p62. Pretreatment with ginsenoside Rb1 inhibited cell viability decrease and increase in doxorubicin-induced autophagic structure and LC3-Ⅱ/LC3-I ratio, and down-regulation of the expression of p62. In conclusion, doxorubicin could induce H9c2 cell death and induce autophagy, and ginsenoside Rb1 showed a protective effect on DOX-induced cardiotoxicity, which may be correlated with suppression of DOX-induced autophagy.

Polysaccharides of Dendrobium officinale Kimura & Migo protect gastric mucosal cell against oxidative damage-induced apoptosis in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendrobium officinale Kimura & Migo (DO) is a valuable Traditional Chinese Medicine to nourish stomach, in which polysaccharides are identified as active ingredients. However, limited scientific evidences have been reported on the gastroprotective efficacy of DO. The aim of the current study was to investigate the protective effects and underlying mechanism of polysaccharides from DO(DOP) on gastric mucosal injury. MATERIAL AND METHODS: For in vitro study, HFE145 cells were pretreated with DOP before induction of cell apoptosis by H2O2. Cell apoptosis and related proteins expression were detected. In the in vivo study, absolute ethanol was administered orally to induce gastric mucosal injury in rat. The gastric mucosal injury area and histological examination were used to evaluate the effects of DOP treatment on the recovery of the gastric mucosal injury. RESULTS: H2O2 treatment for 6h significantly induced cell apoptosis in HFE145 cells. However, the destructive effects of H2O2 on HFE 145 cells could be reversed by the pretreatment with DOP. The increased ROS level induced by H2O2 for 4h was reduced after DOP pretreatment. The number of apoptotic cells in both early and late apoptosis stages decreased significantly and the nuclei morphology changes were improved with DOP pretreatment. Furthermore, DOP inhibited caspase 3 activation and PARP cleavage, downregulated Bax expression and upregulated Bcl2 expression in cell model. Further study revealed that pretreatment of DOP inhibited p -NF-κBp65/NF-κBp65 level, indicating DOP inhibited H2O2-mediated apoptosis via suppression of NF-κB activation. In addition, DOP treatment could ameliorate gastric mucosal injury and inhibit mucin loss induced by ethanol in animal model. DOP treatment also interfered with ethanol-induced apoptosis process by downregulating Bax/Bcl2 ratio in gastric mucosa. CONCLUSIONS: The present study was the first one to demonstrate the gastroprotective effect of DOP through inhibiting oxidative stress-induced apoptosis. This study provided a solid evidence for the potential use of DO as a therapy or health supplement for gastric mucosal diseases.

Vitamin D and Cancer Stem Cells in the Gastrointestinal Tract.

Vitamin D has been widely used as a dietary supplement for the prevention and treatment of bone disorders. Epidemiological and preclinical studies demonstrated the anticancer action of vitamin D in a variety of cancers including those in the gastrointestinal (GI) tract. In these studies the inhibitory action of vitamin D on cancer stem cells (CSCs) has been a focus and is also an important subject to revolutionize the therapeutic potential of vitamin D on cancer treatment. Here, we summarize the involvement of CSC markers and factors and also their signaling pathways in the development of cancers in the esophagus, stomach, colon, pancreas and also liver. It is also evidenced that vitamin D could inhibit these markers and factors and their related signaling pathways to suppress tumor progression. All these information could provide new strategies in repurposing vitamin D as therapeutic agent to inhibit cancers in the GI tract.

In vivo and in vitro anti-tumor and anti-metastasis effects of Coriolus versicolor aqueous extract on mouse mammary 4T1 carcinoma.

Coriolus versicolor (CV), a medicinal mushroom widely consumed in Asian countries, has been demonstrated to be effective in stimulation of immune system and inhibition of tumor growth. The present study aimed to investigate the anti-tumor and anti-metastasis effects of CV aqueous extract in mouse mammary carcinoma 4T1 cells and in 4T1-tumor bearing mouse model. Our results showed that CV aqueous extract (0.125-2 mg/ml) did not inhibit 4T1 cell proliferation while the non-cytotoxic dose of CV extract (1-2 mg/ml) significantly inhibited cell migration and invasion (p<0.05). Besides, the enzyme activities and protein levels of MMP-9 were suppressed by CV extract significantly. Animal studies showed that CV aqueous extract (1 g/kg, orally-fed daily for 4 weeks) was effective in decreasing the tumor weight by 36%, and decreased the lung metastasis by 70.8% against untreated control. Besides, micro-CT analysis of the tumor-bearing mice tibias indicated that CV extract was effective in bone protection against breast cancer-induced bone destruction as the bone volume was significantly increased. On the other hand, CV aqueous extract treatments resulted in remarkable immunomodulatory effects, which was reflected by the augmentation of IL-2, 6, 12, TNF-α and IFN-γ productions from the spleen lymphocytes of CV-treated tumor-bearing mice. In conclusion, our results demonstrated for the first time that the CV aqueous extract exhibited anti-tumor, anti-metastasis and immunomodulation effects in metastatic breast cancer mouse model, and could protect the bone from breast cancer-induced bone destruction. These findings provided scientific evidences for the clinical application of CV aqueous extract in breast cancer patients.

Two immunosuppressive compounds from the mushroom Rubinoboletus ballouii using human peripheral blood mononuclear cells by bioactivity-guided fractionation.

Rubinoboletus ballouii is an edible mushroom wildly grown in Yunnan province, China. Up till now, little was known about the chemical and biological properties of this mushroom. The aim of this study was to investigate the immunomodulatory effects of the ethanolic extract of Rubinoboletus ballouii and its fractions on human peripheral blood mononuclear cells (PBMCs) using bioactivity-guided fractionation. The crude extract of the fruiting bodies of RB was fractionated by high-speed counter current chromatography (HSCCC). Twelve fractions were obtained and the third fraction (Fraction C) exerted the most potent anti-inflammatory activities in mitogen-activated PBMCs. Further fractionation of fraction C led to the isolation of two single compounds which were elucidated as 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin, respectively. The results showed that both 1-ribofuranosyl-s-triazin-2(1H)-one and pistillarin exhibited significant immunosuppressive effects on phytohemagglutinin (PHA)-stimulated human PBMCs by inhibiting [methyl-(3)H]-thymidine uptake and inflammatory cytokines productions such as tumor necrosis factor (TNF)-α, interleukin (IL)-10, interferon (IFN)-γ and IL-1ß. Besides, 1-ribofuranosyl-s-triazin-2(1H)-one was firstly found in natural resources, and pistillarin was also isolated from the family Boletaceae for the first time. They exhibited great potential in developing as anti-inflammatory reagents.