Aucubin administration suppresses STING signaling and mitigated high-fat diet-induced atherosclerosis and steatohepatosis in LDL receptor deficient mice.

The rising obesity epidemic in developed countries is associated with many chronic inflammatory diseases including atherosclerosis and nonalcoholic steatohepatitis (NASH). Consuming aucubin may benefit health by suppressing inflammation. Herein, we studied the effects of aucubin consumption on atherosclerosis and NASH progression induced by high-fat diet (HFD) in LDL receptor deficient (LDLr-/-) mice. Adult LDLr-/- mice were fed with HFD for 12 weeks and received oral administration of aucubin for the last 6 weeks. Aucubin did not alter body weight or dyslipidemia, but lowered hyperglycemia and mitigated HFD-induced atherosclerosis and hepatic impairments in LDLr-/- mice. Aucubin administration inhibited HFD-induced inflammation and downregulated mRNA and protein expression of stimulator of IFN genes (STING) in both aortas and livers of LDLr-/- mice. In vitro, aucubin suppressed mitochondrial DNA (mtDNA)-induced activation of STING/NFκB pathway and downregulated gene expression of pro-inflammatory cytokines in cultured bone marrow-derived macrophages (BMDM). Furthermore, aucubin enhanced microRNA-181a-5p (miR-181a-5p) levels in both aortas and livers of LDLr-/- mice. Importantly, miR-181a-5p mimicked the inhibitory effect of aucubin on STING/NFκB pathway and inflammation in BMDM. In conclusion, aucubin consumption attenuated HFD-induced atherosclerosis and NASH progression in LDLr-/- mice, possibly through modulating miR-181a-5p/STING and inhibiting inflammation.

Aucubin slows the development of osteoporosis by inhibiting osteoclast differentiation via the nuclear factor erythroid 2-related factor 2-mediated antioxidation pathway.

CONTEXT: Osteoporosis (OP) is a metabolic disease. We have previously demonstrated that aucubin (AU) has anti-OP effects that are due to its promotion of the formation of osteoblasts. OBJECTIVES: To investigate the mechanisms of anti-OP effects of AU. MATERIALS AND METHODS: C57BL/6 mice were randomly divided into control group, 30 mg/kg Dex-induced OP group (OP model group, 15 µg/kg oestradiol-treated positive control group, 5 or 45 mg/kg AU-treated group), and 45 mg/kg AU-alone-treated group. The administration lasted for 7 weeks. Subsequently, 1, 2.5 and 5 µM AU were incubated with 50 ng/mL RANKL-induced RAW264.7 cells for 7 days to observe osteoclast differentiation. The effect of AU was evaluated by analysing tissue lesions, biochemical factor and protein expression. RESULTS: The LD50 of AU was greater than 45 mg/kg. AU increased the number of trabeculae and reduced the loss of chondrocytes in OP mice. Compared to OP mice, AU-treated mice exhibited decreased serum concentrations of TRAP5b (19.6% to 28.4%), IL-1 (12.2% to 12.6%), IL-6 (12.1%) and ROS (5.9% to 10.7%) and increased serum concentrations of SOD (14.6% to 19.4%) and CAT (17.2% to 27.4%). AU treatment of RANKL-exposed RAW264.7 cells decreased the numbers of multi-nuclear TRAP-positive cells, reversed the over-expression of TRAP5, NFATc1 and CTSK. Furthermore, AU increased the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream proteins in RANKL-exposed RAW264.7 cells. CONCLUSIONS: AU slows the development of OP via Nrf2-mediated antioxidant pathways, indicating the potential use of AU in OP therapy and other types of OP research.

Swertiamarin supplementation prevents obesity-related chronic inflammation and insulin resistance in mice fed a high-fat diet.

Obesity is characterized by low-grade chronic inflammation, which underlies insulin resistance and non-alcoholic fatty liver disease (NAFLD). Swertiamarin is a secoiridoid glycoside that has been reported to ameliorate diabetes and NAFLD in animal models. However, the effects of swertiamarin on obesity-related inflammation and insulin resistance have not been fully elucidated. Thus, this study investigated the effects of swertiamarin on inflammation and insulin resistance in high-fat diet (HFD)-induced obese mice. C57BL/6 mice were fed a HFD or HFD containing swertiamarin for 8 weeks. Obesity-induced insulin resistance and inflammation were assessed in the epididymal white adipose tissue (eWAT) and livers of the mice. Swertiamarin attenuated HFD-induced weight gain, glucose intolerance, oxidative stress, and insulin resistance, and enhanced insulin signalling in mice. Compared to HFD-fed mice, the swertiamarin-treated mice exhibited increased lipolysis and reduced adipocyte hypertrophy and macrophage infiltration in eWAT. Moreover, swertiamarin alleviated HFD-mediated hepatic steatosis and inflammation by suppressing activation of the p38 MAPK and NF-κB pathways within the eWAT and liver of obese mice. In conclusion, supplementation with swertiamarin attenuated weight gain and hepatic steatosis, and alleviated obesity-associated inflammation and insulin resistance, in obese mice.

Detection and Identification of Catalpol Metabolites in the Rat Plasma, Urine and Faeces Using Ultra-high Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-orbitrap High-resolution Accurate Mass Spectrometry.

BACKGROUND: Catalpol, an iridoid glycoside, is one of the richest bioactive components present in Rehmannia glutinosa. More and more metabolites of drugs have exhibited various pharmacological effects, thus providing guidance for clinical application. However, few researches have paid attention to the metabolism of catalpol. OBJECTIVE: This study aimed to establish a rapid and effective method to identify catalpol metabolites and evaluate the biotransformation pathways of catalpol in rats. METHODS: In this study, catalpol metabolites in rat urine, plasma and faeces were analyzed by UHPLC-Q-Exactive MS for the characterization of the metabolism of catalpol. Based on high-resolution extracted ion chromatograms (HREICs) and parallel reaction monitoring mode (PRM), metabolites of catalpol were identified by comparing the diagnostic product ions (DPIs), chromatographic retention times, neutral loss fragments (NLFs) and accurate mass measurement with those of catalpol reference standard. RESULTS: A total of 29 catalpol metabolites were detected and identified in both negative and positive ion modes. Nine metabolic reactions, including deglycosylation, hydroxylation, dihydroxylation, hydrogenation, dehydrogenation, oxidation of methylene to ketone, glucuronidation, glycine conjugation and cysteine conjugation, were proposed. CONCLUSION: A rapid and effective method based on UHPLC-Q-Exactive MS was developed to mine the metabolism information of catalpol. Results of metabolites and biotransformation pathways of catalpol suggested that when orally administrated, catalpol was firstly metabolized into catalpol aglycone, after which phase I and phase II reactions occurred. However, hydrophilic chromatography-mass spectrometry is still needed to further find the polar metabolites of catalpol.

Protective effects of catalpol on mitochondria of hepatocytes in cholestatic liver injury.

Cholestasis, which is caused by the obstruction of bile flow, can lead to rapid organ injury, cell apoptosis and necrosis of hepatocytes, and may eventually develop into fibrosis and cirrhosis. Oxidative stress and mitochondrial dysfunction are the key pathogenic signs of hepatic cholestasis. Catalpol has pharmacological activities, including antioxidative and anti­inflammatory effects, and may relieve mitochondrial damage and restore mitochondrial membrane potential. However, the potential roles and mechanisms of catalpol in cholestasis­induced liver injury are not clear. In the present study, liver function­related indexes were measured in the serum of mice by commercial kits. In addition, levels of serum inflammatory factors were detected by ELISA. Hematoxylin and eosin staining was performed to observe histopathological changes, and mitochondrial membrane potential was detected using JC­1 staining. Mitochondrial adenosine triphosphate (ATP), reactive oxygen species (ROS) and malondialdehyde levels were determined using a luciferase reporter kit, flow cytometry and a thiobarbituric acid reactive substance assay kit, respectively. Western blotting was performed to detect the expression levels of apoptosis­related proteins in liver tissues. The findings revealed that catalpol reduced liver damage caused by cholestasis, improved the mitochondrial membrane potential, and increased the ATP content and glutathione content of cholestasis model mice. Moreover, catalpol also reduced the ROS level, inhibited lipid peroxidation, and regulated oxidative stress and apoptotic protein expression. Thus, the present study preliminarily confirmed that catalpol can reduce liver injury in a mouse model of cholestasis through inhibiting oxidative stress and enhancing mitochondrial membrane potential.

Aucubin administered by either oral or parenteral route protects against cisplatin-induced acute kidney injury in mice.

Aucubin is pharmacologically active natural compound which possesses numerous beneficial properties. This study aimed to evaluate the protective effect of aucubin against cisplatin (CP)-induced acute kidney injury in mice and the mechanism of its action. Aucubin was administrated to mice orally or intraperitoneally (ip) (1.5 and 5 mg/kg) for two consecutive days, two days after ip injection of cisplatin (CP), 11 mg/kg. Treatment with aucubin by both routes of administration ameliorated histopathological changes and reduced elevated serum markers of kidney injury. CP administration increased renal expression of heme oxygenase-1 (HO-1) and 4-hydroxynonenal (4-HNE), as well as tumor necrosis factor-alpha (TNF-α), which was dose-dependently ameliorated by aucubin. Moreover, aucubin reduced increased renal expression of cleaved caspase-3 and -9 and decreased poly (ADP-ribose) polymerase (PARP) cleavage. Mechanistically, aucubin suppressed the activation of several signaling pathways involved in inflammation and apoptosis, including nuclear factor-kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), Akt, extracellular signal-regulated kinase 1/2 (ERK1/2) and forkhead box O3a (FOXO3a). Parenteral application was marginally but statistically more effective in reducing CP-induced kidney injury than oral administration. The findings of this study suggest that aucubin acts as a protective agent against CP-induced nephrotoxicity, which should be further investigated.

Determination of aucubin by supramolecular solvent-based dispersive liquid-liquid microextraction and UPLC-MS/MS: Application to a pharmacokinetic study in rats with type 1 diabetes.

A novel method was developed for the determination of aucubin in rat serum by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with supramolecular solvent (SUPRAS)-based dispersive liquid-liquid microextraction (DLLME). The SUPRAS was prepared with pentanol as extraction solvent and tetrahydrofuran as dispersing agent. Based on single factor test and response surface methodology, critical parameters were optimized as: 1 mL of pentanol as extraction solvent, 4 mL of tetrahydrofuran as dispersing agent, 200 µL of SUPRAS, and vortex duration of 2.5 min. The established method was validated in terms of selectivity, linearity, accuracy, precision, recovery, stability, and applied to a pharmacokinetic study on type 1 diabetes model rats intraperitoneally administered with aucubin. The experimental results showed that the maximum concentration in serum (Cmax) and area under the serum concentration versus time curve (AUC) of aucubin in type 1 diabetic rats were higher than those in normal rats. Such pharmacokinetic alterations might presumably result from the pathological state of type 1 diabetes. The current study may provide scientific evidence and inspiring insights for clinical application of aucubin for the treatment of diabetes.

Sweroside Prevents Non-Alcoholic Steatohepatitis by Suppressing Activation of the NLRP3 Inflammasome.

Non-alcoholic steatohepatitis (NASH), a type of non-alcoholic fatty liver disease, is characterized as steatosis and inflammation in the liver. NLRP3 inflammasome activation is associated with NASH pathology. We hypothesized that suppressing the NLRP3 inflammasome could be effective in preventing NASH. We searched substances that could inhibit the activation of the NLRP3 inflammasome and identified sweroside as an NLRP3 inhibitor. We investigated whether sweroside can be applied to prevent the pathological symptoms associated with NASH in a methionine-choline-deficient (MCD) diet-induced NASH mouse model. The activation of the NLRP3 inflammasome was determined by detecting the production of caspase-1 and IL-1ß from pro-caspase-1 and pro-IL-1ß in primary mouse macrophages and mouse liver. In a NASH model, mice were fed an MCD diet for two weeks with daily intraperitoneal injections of sweroside. Sweroside effectively inhibited NLRP3 inflammasome activation in primary macrophages as shown by a decrease in IL-1ß and caspase-1 production. In a MCD diet-induced NASH mouse model, intraperitoneal injection of sweroside significantly reduced serum aspartate transaminase and alanine transaminase levels, hepatic immune cell infiltration, hepatic triglyceride accumulation, and liver fibrosis. The improvement of NASH symptoms by sweroside was accompanied with its inhibitory effects on the hepatic NLRP3 inflammasome as hepatic IL-1ß and caspase-1 were decreased. Furthermore, sweroside blocked de novo synthesis of mitochondrial DNA in the liver, contributing to suppression of the NLRP3 inflammasome. These results suggest that targeting the NLRP3 inflammasome with sweroside could be beneficially employed to improve NASH symptoms.

Loganin and catalpol exert cooperative ameliorating effects on podocyte apoptosis upon diabetic nephropathy by targeting AGEs-RAGE signaling.

BACKGROUND/AIMS: Rehmanniae Radix (RR) and Cornus officinalis (CO) are a typical herbal pair used to treat diabetic nephropathy (DN) in clinical practice. DN can be effectively treated by catalpol (Cat) and loganin (Log), the main active components of RR and CO respectively, through combating apoptosis, oxidative stress and inflammation. Herein, a spontaneous DN and podocyte injury model induced by advanced glycation end products (AGEs), i.e. KK-Ay mice, was used to explore the cooperative effects of Log and Cat on DN and the mechanism targeting the AGEs-RAGE (receptor for AGE) pathway. METHODS AND KEY FINDINGS: Log and Cat alone or in combination mitigated diabetic symptoms, decreased the level of fasting blood glucose, and increased that of serum insulin. The two drugs alone or in combination protected renal function from damage, prevented extracellular matrix hyperplasia and glycogen deposition, as well as alleviated the loss of podocytes detected by histological assay and immunohistochemistry. Flow cytometry revealed that Log and Cat alone or in combination relieved the apoptosis of AGEs-induced podocytes in vitro. Silencing RAGE by RNA interference played a protective role in podocyte apoptosis, whereas overexpression of it worked oppositely. Western blot exhibited that Log and Cat alone or in combination inhibited the activation of RAGE/p38 MAPK/p65 NF-κB and RAGE/Nox4/p65 NF-κB pathways in podocytes. The inhibitory effects of drug combination were more evident than those of individual treatments. SIGNIFICANCE: Log and Cat cooperatively resisted the apoptosis of podocytes upon DN by targeting AGEs-RAGE and its downstream pathways p38 MAPK and Nox4.

The hypoglycemic mechanism of catalpol involves increased AMPK-mediated mitochondrial biogenesis.

Mitochondria serve as sensors of energy regulation and glucose levels, which are impaired by diabetes progression. Catalpol is an iridoid glycoside that exerts a hypoglycemic effect by improving mitochondrial function, but the underlying mechanism has not been fully elucidated. In the current study we explored the effects of catalpol on mitochondrial function in db/db mice and C2C12 myotubes in vitro. After oral administration of catalpol (200 mg·kg-1·d-1) for 8 weeks, db/db mice exhibited a decreased fasting blood glucose level and restored mitochondrial function in skeletal muscle. Catalpol increased mitochondrial biogenesis, evidenced by significant elevations in the number of mitochondria, mitochondrial DNA levels, and the expression of three genes associated with mitochondrial biogenesis: peroxisome proliferator-activated receptor gammaco-activator 1 (PGC-1α), mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1). In C2C12 myotubes, catalpol significantly increased glucose uptake and ATP production. These effects depended on activation of AMP-activated protein kinase (AMPK)-mediated mitochondrial biogenesis. Thus, catalpol improves skeletal muscle mitochondrial function by activating AMPK-mediated mitochondrial biogenesis. These findings may guide the development of a new therapeutic approach for type 2 diabetes.

Catalpol ameliorates Sjögren's Syndrome by modulating interplay of T and B cells.

Catalpol is an active ingredient of Rehmanniae Radix, a medical herb used frequently in treating primary Sjogren's Syndrome (pSS). However, no study has assessed the therapeutic effects of catalpol in treating Sjogren's Syndrome. This study aimed to investigate the therapeutic effects of catalpol for pSS by evaluating the water consumption, stimulated salivary flow rates, sialadenitis, T cell subsets and related cytokine levels. Catalpol treated mice had improved stimulated salivary flow rates and water consumption compared to mice from the control group. Catalpol also reduced the lymphocytic infiltration and prevented the formation of ectopic germinal centers. These effects, especially in the catalpol high dose group, were associated with elevated CD4+ CXC-R5+ PD-1+ Foxp3+ T follicular regulatory (Tfr) cells (1.572 % vs 1.118 %, P = 0.0005) and a higher ratio of Tfr cells to T follicular helper (Tfh) cells (2.137 vs 1.541, P = 0.0007). Downregulated levels of IFN-γ and BAFF in serum and submandibular glands were also noted in catalpol treated groups. Our work indicated that catalpol might be a potential drug for treating pSS by regulating the interplay between T and B cells.

Simultaneous determination of multiple components in rat plasma and pharmacokinetic studies at a pharmacodynamic dose of Xian-Ling-Gu-Bao capsule by UPLC-MS/MS.

Xian-Ling-Gu-Bao capsule (XLGB) is an effective traditional Chinese medicine prescription (TCMP) that is used for the prevention and treatment of osteoporosis in China. A rapid, simple, efficient and stable method based on UPLC-MS/MS technology was developed for simultaneous determination of multiple components of XLGB in rat plasma. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode with electrospray ionization (ESI). For twenty-one selected quantitative prototypes, all calibration curves showed favourable linearity (r>0.9932) in linear ranges. The lower limits of quantification (LLOQs) were 2 ng/mL for psoralen (PL), 2.5 ng/mL for asperosaponin VI (AS), 1 ng/mL for isopsoralen (IPS) and sweroside (SW), 0.5 ng/mL for magnoflorine (MA), bavachinin (BVN), tanshinone IIA (TA), timosaponin BII (TBII) and icaritin (ICT), 0.1 ng/mL for epimedin B (EB) and epimedin C (EC), 0.05 ng/mL for icariin (IC), isobavachalcone (IBC), psoralidin (PD), bavachin (BV), bavachalcone (BC), epimedin A (EA) and isobavachin (IBV), 0.02 ng/mL for neobavaisoflavone (NEO) and icariside I (ICI) and 0.01 ng/mL for icariside II (ICII). The intra-day and inter-day (low, medium, high) precision (relative standard deviation) for all analytes was less than 8.63%, and the accuracies (as relative error) were in the range of -12.45% to 8.91%. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The validated method was successfully applied to the pharmacokinetics (PK) studies of the twenty-one prototypes at pharmacodynamic doses (0.3 and 1 g/kg/day). In addition, dynamic profiles of 28 metabolites (phase II conjugates: 23 glucuronide conjugates, 2 sulfate conjugates and 3 glucuronide or sulfate conjugates) were also monitored by their area/IS area-time curves. As a result, coumarins, prenylated flavonoids from Psoraleae Fructus, alkaloids and prenylated flavonol glycosides from Epimedii Herba, and iridoid glycosides, triterpenoid saponins from Dipsaci Asperoidis Radix were considered to be the key effective substances of XLGB due to their high exposure and appropriate pharmacokinetic features. This is the first report to reveal pharmacodynamic ingredients by a reversed pharmacodynamic (PD) - pharmacokinetics (PK) study.

Oleuropein-enriched chocolate by extra virgin olive oil blunts hyperglycaemia in diabetic patients: Results from a one-time 2-hour post-prandial cross over study.

BACKGROUND & AIMS: Oleuropein, a component of extra virgin olive oil (EVOO), reduces post-prandial glycemia with a mechanism counteracting oxidative stress-mediated incretin down-regulation. In this study we evaluated if the intake of an oleuropein-enriched chocolate could have positive effects on glycaemia and insulin levels in patients with type 2 diabetes mellitus (T2DM) and healthy subjects (HS). METHODS: Twenty-five consecutive T2DM patients and 20 HS were recruited. Participants were randomized to receive 40 g oleuropein-enriched chocolate by EVOO or 40 g control chocolate spread in a cross-over design. Serum glucose, insulin, glucagon-like peptide-1 (GLP1), and dipeptidyl-peptidase-4 (DPP4) were measured before and 2 h after chocolate intake. RESULTS: In T2DM, the pairwise comparisons showed that intake of oleuropein-enriched chocolate was associated with a significantly less increase of blood glucose compared to control; GLM analysis showed a significant difference for treatments with respect to glucose (p = 0.04), GLP1 (p < 0.001) and DPP-4 activity (p = 0.01). In HS, the pairwise comparisons showed that, after oleuropein-enriched chocolate intake, blood glucose concentration and DPP4 activity did not change; conversely a significant increase was observed for insulin and GLP1. After control chocolate intake, a significant increase for blood glucose, insulin levels and DPP4 activity were observed while GLP1 did not change. CONCLUSION: The study shows that using EVOO as source of oleuropein administration of 40 g oleuropein-enriched chocolate is associated with a modest increase or no change of glycemia in T2DM and HS respectively, via an incretin-mediated mechanism.

Pharmacokinetics and metabolic profiles of swertiamarin in rats by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

Swertiamarin, a typical compound of secoiridiod glycosides with various pharmacological effects which is the major iridoid glicoside of Swertia. In this study, we have established a fast and sensitive LC-MS/MS method. The aim was to conduct pharmacokinetic studies of swertiamarin in vivo of rats. Gentiopicroside was used as internal standard and a C18 column was employed for the separation of analytes. The selected reaction monitoring transitions were m/z 375→177, 357.1→195 for swertiamarin and the internal standard, respectively, in a positive ion mode. The results showed that swertiamarin had a good linearity in the range of 2-8000 ng/mL (r ＞ 0.997) and its limit of detection (LLOD) was 0.5 ng/mL. The developed method subsequently successfully used in the pharmacokinetic study of swertiamarin in rats after oral administration (50, 100, and 150 mg/kg). We obtained a series of pharmacokinetic parameters, and the half-time of swertiamarin was 1 h, while the oral bioavailability was between 5.6-7.6%. Six metabolites of swertiamarin were identified based on accurate mass measurements of protonated molecules and their MS/MS spectrum by ultra-high-performance chromatography/tandem quadrupole time-of-flight mass spectrometry. Furthermore, metabolites were classified into three groups and the metabolic pathway of swertiamarin was proposed. The finding may help for the understanding of effectiveness and safety of swertiamarin.

Catalpol Enhances Neurogenesis And Inhibits Apoptosis Of New Neurons Via BDNF, But Not The BDNF/Trkb Pathway.

BACKGROUND: The role of catalpol in brain neurogenesis and newborn neuron survival has not been previously determined in permanent middle cerebral artery occlusion (pMCAO). METHODS: Fifty-four rats were divided into 6 groups: pMCAO (model, n=9); sham operation (NS, n=9); catalpol treatment (5 mg/kg and 10 mg/kg subgroups, n=9 each); K252a (n=9); and K252a+catalpol 5 mg/kg (n=9) with stroke. The effects of catalpol on behavior, neurogenesis surrounding the infarction ipsilateral to pMCAO, and the expression of brain-derived neurotrophic factor (BDNF) and its receptor (TrkB) were evaluated. Vehicle or, K252a (i.p.), an inhibitor of TrkB phosphorylase. RESULTS: Repeated administration of catalpol reduced neurological deficits and significantly improved neurogenesis. Catalpol increased the number of newborn immature neurons, as determined by BrdU+-Nestin+ and BrdU+-Tuj-1+ staining, and downregulated cleaved caspase 3 in Tuj-1+ cells at day 7 following stroke. Moreover, catalpol increased the protein expression of Tuj-1, MAP2, and the Bcl-2/Bax ratio, as determined using Western blot. Catalpol also significantly increased brain levels of BDNF, but not TrkB, resulting in enhanced survival of newborn neurons via inhibition of apoptosis. CONCLUSION: Catalpol may contribute to neurogenesis in infarcted brain regions and help promote the survival of newborn neurons by activating BDNF, but not BDNF/TrkB signaling.

Gentiopicrin exerts anti-rheumatic effect in human fibroblast-like synoviocytes via inhibition of p38MAPK/NF-κB pathway.

To investigate the effect of gentiopicrin on the expressions of inflammatory factors in human fibroblast-like synoviocytes (HFLS) and the underlying mechanism. Human fibroblast-like synoviocytes (HFLS) were cultured in vitro at 37 °C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5 % fetal bovine serum (FBS) in a humidified incubator containing 5 % CO2. Cell viability was determined using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide (MTT) assay, while real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expressions of interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) mRNAs. The expressions of p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB) were determined using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IL-1ß and IL-6 in cell lysate. Treatment with 5-25 µM gentiopicrin did not significantly affect the number of viable cells, when compared with control group (p > 0.05). However, at 50 and 100 µM gentiopicrin, the number of viable cells were significantly increased, relative to control group (p < 0.05). Results of qRT-PCR showed that the expression levels of IL-1ß and IL-6 mRNAs were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently decreased their expression levels compared with TNF-α group (p < 0.05). Western blotting results showed that the expressions of p-p38MAPK and NF-κB-p65 proteins were significantly upregulated in TNF-α group, when compared with control group (p < 0.05). However, treatment with gentiopicrin significantly and dose-dependently down-regulated the expression of these proteins compared with TNF-α group (p < 0.05). The levels of IL-1ß and IL-6 in cell lysate were significantly higher in TNF-α group than in control group (p < 0.05). However, treatment with gentiopicrin, and p38MAPK/NF-κB pathway inhibitors (SB203580 and BAY11-7082) significantly reduced the levels of these inflammatory factors compared with TNF-α group (p < 0.05).  Gentiopicrin has therapeutic potential for Rheumatoid arthritis (RA  ) through a mechanism involving the inhibition of p38MAPK/NF-κB pathway.

Catalpol alleviates adriamycin-induced nephropathy by activating the SIRT1 signalling pathway in vivo and in vitro.

BACKGROUND AND PURPOSE: Catalpol, a water-soluble active ingredient isolated from Rehmannia glutinosa, exhibits multiple pharmacological activities. However, the mechanism(s) underlying protection against renal injury by catalpol remains unknown. EXPERIMENTAL APPROACH: Adriamycin-induced kidney injury models associated with podocyte damage were employed to investigate the nephroprotective effects of catalpol. In vivo, TUNEL and haematoxylin-eosin staining was used to evaluate the effect of catalpol on kidney injury in mice. In vitro, effects of catalpol on podocyte damage induced by adriamycin was determined by elisa kit, flow cytometry, Hoechst 33342, and TUNEL staining. The mechanism was investigated by siRNA, EX527, and docking simulations. KEY RESULTS: In vivo, catalpol treatment significantly improved adriamycin-induced kidney pathological changes and decreased the number of apoptotic cells. In vitro, catalpol markedly decreased the intracellular accumulation of adriamycin and reduced the calcium ion level in podocytes and then attenuated apoptosis. Importantly, the regulatory effects of catalpol on sirtuin 1 (SIRT1), multidrug resistance-associated protein 2 (MRP2), and the TRPC6 channel were mostly abolished after incubation with SIRT1 siRNA or the SIRT1-specific inhibitor EX527. Furthermore, docking simulations showed that catalpol efficiently oriented itself in the active site of SIRT1, indicating a higher total binding affinity score than that of other SIRT1 activators, such as resveratrol, SRT2104, and quercetin. CONCLUSION AND IMPLICATIONS: Taken together, our results suggest that catalpol exhibits strong protective effects against adriamycin-induced nephropathy by inducing SIRT1-mediated inhibition of TRPC6 expression and enhancing MRP2 expression.

Sweroside-mediated mTORC1 hyperactivation in bone marrow mesenchymal stem cells promotes osteogenic differentiation.

This paper aims to probe into the effect of sweroside (SOS) in osteoporosis (OP) and explains mechanisms of its molecular. Applying the ovariectomized (OVX) mouse model investigates the preventive effect of SOS against postmenopausal OP after 3 months of SOS treatment (120 mg/kg/day). Using hematoxylin and eosin (HE) staining and micro computed tomography (CT) observed the morphology of OP in each group. Immunohistochemical staining (IHC) was used to examine osteoblast markers. Experiments in vitro, bone marrow mesenchymal stem cells (BMSCs) from C57/BL6 mice were treated with SOS for 14 days. The staining of alizarin red and alkaline phosphatase activity were measured, and the presentation of osteoblast markers was detected by quantitative reverse transcription PCR. BMSCs were also treated with 1 µg/mL SOS with or without rapamycin, the expression of protein S6 (PS6), P-mTOR, runt-related transcription factor 2 (RUNX2), OSX, and osteocalcin (OCN) was detected by Western blotting. Experiments in vivo, HE results show that SOS can alleviate OP, CT results show that there are lower trabecular thickness, bone mineral density, and trabecular number in control OVX mice than those in the OVX + SOS group. IHC results showed that SOS can promote the expression of osteogenic markers and immunofluorescent results show that SOS can promote mTORC1 signal activation. Experiments in vitro revealed that SOS stimulated the activation of the mTORC1 signaling pathway and upregulated RUNX2, OSX, and OCN, rapamycin can reverse it. Our findings demonstrated that differentiated BMSCs into osteoblasts can be promoted by SOS via upregulating the expression of P-mTOR, PS6, RUNX2, OSX, and OCN. SOS effectively prevented OP by hyperactivation of the mTORC1/PS6 signaling pathway.

Sweroside Alleviated LPS-Induced Inflammation via SIRT1 Mediating NF-κB and FOXO1 Signaling Pathways in RAW264.7 Cells.

Pterocephalus hookeri was used as a traditional Chinese medicine for the treatment of rheumatoid arthritis. Sweroside was a main iridoid isolated from P. hookeri. The present study aimed to investigate the anti-inflammatory effect mechanism of sweroside. In RAW264.7 cells induced by lipopolysaccharide (LPS), the abnormal proliferation, the NO content increase, and the downregulated Sirtuin1 (SIRT1) expression were observed. Sweroside could alleviate the inflammation by inhibiting cell proliferation through arresting the cell cycle at the G0/G1 phase, by suppressing pro-inflammatory cytokines and by promoting anti-inflammatory cytokines in LPS-induced RAW264.7 cells. Further mechanism research indicated that sweroside could activate the SIRT1, then suppress the nuclear factor-kappa B (NF-κB) and promote the Forkhead transcription factor O1 (FOXO1) signaling pathways. The present study indicated that sweroside may be the main anti-inflammatory constituent of P. hookeri and a promising candidate for anti-inflammation therapy.

The therapeutic effect of aucubin-supplemented hyaluronic acid on interleukin-1beta-stimulated human articular chondrocytes.

BACKGROUND: Injection of exogenous hyaluronic acid (HA) into the joint capsule improves symptoms of early stage osteoarthritis (OA). However, reactive oxygen species degrade HA into small oligosaccharides that can elicit pro-inflammatory responses. Likewise, disturbance of the antioxidant enzyme system and generation of oxidative stress by pro-inflammatory cytokines worsen knee OA. Accordingly, we proposed the use of aucubin, an antioxidant and anti-inflammatory compound, as a versatile adjuvant to HA for treating OA. METHODS: Primary human chondrocytes were cultured in media supplemented with aucubin in a series of concentrations (0, 0.01, 0.1, 1, and 10 µg/ml) to study dose-dependent toxicity. We then evaluated the therapeutic effects of HA (100 µg/ml) supplemented with aucubin (10 µg/ml) on interleukin-1 beta (IL-1ß, 10 ng/ml)-stimulated chondrocytes. RESULTS: The use of aucubin did not change cell viability or alter lactate dehydrogenase release to normal chondrocytes. Although the proliferation and sulfated glycosaminoglycan production were not affected, aucubin partially restored the hypertrophic transformation of chondrocytes. Relative to treatment with HA or aucubin alone, real-time PCR revealed that aucubin-supplemented HA down-regulated the mRNA levels of tumor necrosis factor-alpha (TNF-α), corrected collagen type 1 and aggrecan, and up-regulated tissue inhibitor of metalloproteinase 1. Moreover, ELISA testing also showed a reduced TNF-α production. Although superoxide dismutases activity was still distributed, aucubin restored total antioxidant capacity of IL-1ß-stimulated chondrocytes. Western blotting further showed that aucubin inhibited cyclooxygenase-2 and regulated the nuclear factor (erythroid-derived 2)-like 2 pathway. CONCLUSION: Aucubin can enhance the anti-catabolic and anti-inflammatory effects of HA on OA chondrocytes.