Anti-osteoporosis effect of Semen Cuscutae in ovariectomized mice through inhibition of bone resorption by osteoclasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Cuscutae, called Tu-si-zi in Chinese, is a kind of dried mature seed in the Convolvulaceae family. It mainly distributes in China, Korea, Pakistan, Vietnam, India and Thailand. It is used as a kidney-tonifying drug for treatment of aging related diseases such as osteoporosis in traditional Chinese medicine. However, the exact mechanisms on bone resorption are poorly studied. AIM OF THE STUDY: The aim of this study was to investigate the potential effect of Semen Cuscutae on ovariectomy (OVX)-induced osteoporosis in mice and clarify the exact mechanisms by which Semen Cuscutae exert the anti-osteoporosis effect. MATERIALS AND METHODS: Qualitative and quantitative analyses of Semen Cuscutae were performed by UPLC-Q-TOF-MS and HPLC-MS/MS, respectively. Changes in bone mineral density (BMD) induced by OVX in mice were measured by dual-energy X-ray absorptiometry and micro-computed tomography (µCT). Tartrate-resistant acid phosphatase (TRAP) staining as well as hematoxylin and eosin (HE) staining were used to observe bone microarchitectural changes. ELISA kits were used to assess the therapeutic effects of Semen Cuscutae on the serum levels of osteoprotegerin (OPG), tartrate-resistant acid phosphatase 5b (TRACP-5b), and receptor activator of nuclear factor-κB (RANKL). The effect of Semen Cuscutae on primary cell viability was assessed using CCK-8 and anti-tartrate phosphatase assays. TRAP staining and actin ring staining were used to observe the effect of Semen Cuscutae on osteoclast differentiation. Western blotting was used to measure the effects of Semen Cuscutae on expressions of NFATC1, c-Src kinase, and c-fos. RESULTS: Results from UPLC-Q-TOF-MS showed that the main components of Semen Cuscutae were flavonoid compounds that included quercitrin, quercetin, hyperoside, caffeic acid, rutin, chlorogenic acid, luteolin, apigenin, kaempferol, isoquercetin, cryptochlorogenic acid, isorhamnetin-3-O-glucoside, and astragalin. After the Semen Cuscutae extract was orally administered to OVX mice, bone density increased (P < 0.01) and bone microstructure was significantly improved (P < 0.01 or 0.05). Additionally, Semen Cuscutae exhibited a significant descending effect in the levels of serum TRACP-5b and RANKL, while there was a significant increase in OPG in the Semen Cuscutae group compared with the OVX group, especially at high doses. Moreover, we found that increasing of c-fos, c-Src kinase, and NFATC1 protein expressions were reversed by Semen Cuscutae in vitro and in vivo. CONCLUSIONS: Our results showed that Semen Cuscutae exhibited anti-osteoporosis effects through the c-fos/c-Src kinase/NFATC1 signaling pathway.

Anti-perimenopausal osteoporosis effects of Erzhi formula via regulation of bone resorption through osteoclast differentiation: A network pharmacology-integrated experimental study.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi formula (EZF) consists of Ecliptae herba (EH) and Fructus Ligustri Lucidi (FLL) at a ratio 1:1, and constitutes a well-known formula in China that is commonly used for treating menopausal diseases. AIM OF THE STUDY: In this study, we explored the pharmacologic actions and potential molecular mechanisms underlying EZF's action in preventing and treating osteoporosis. MATERIALS AND METHODS: The active components and related targets of EZF's anti-osteoporotic effects were predicted by network pharmacology, and functional enrichment analysis was also performed. We then used an osteoporosis model of ovariectomized (OVX) mice to detect the effects of EZF on osteoporosis. RESULTS: The results from network pharmacology identified a total of 10 active ingredients from EH and 13 active ingredients from FLL that might affect 65 potential therapeutic targets. GO enrichment analysis revealed that EZF affected bone tissue primarily via hormone (particularly estradiol)-related pathways and bone resorption by osteoclast differentiation. KEGG analysis demonstrated that bone-related factors such as Runt-related transcription factor 2 (Runx2), Ca2, estrogen receptor1 (ESR1), androgen receptors (AR), and TNFα served as the primary targets during osteoclastic differentiation. In vivo experiments showed that the formula significantly improved the diminution in estrogen and the subsequent uterine atrophy induced by ovariectomy (P < 0.01 or 0.05), implying that the EZF exerted its actions via regulation of estradiol and the nourishing effects of the uterus in OVX mice. Dual-energy X-ray absorptiometry and micro-CT showed that EZF significantly inhibited bone loss and improved bone micro-architecture by statistically increasing the number of bone trabeculae and decreasing the separation of bone trabeculae in OVX mice (P < 0.01 or 0.05); EZF also inhibited bone loss and enhanced bone-fracture load. Furthermore, we confirmed that EZF reduced the calcium concentrations, augmented protein and mRNA levels for Runx2 in the bone marrow, and reduced PPARγ levels. RANKL-a key downstream regulatory protein of many targets that was referred to in our results of network pharmacology as being involved in the regulation of osteoclastogenesis-was significantly diminished by EZF; it also elevated OPG content. In addition, we used monocytes of bone-marrow origin to detect the effects of the potential components of EZF on osteoclast differentiation and found that wedelolactone, oleanolic acid, echinocystic acid, luteolin, and luteolin-7-o-glucoside significantly inhibited osteoclast differentiation from monocytes induced by 25 ng/mL MCSF and 50 ng/mL RANKL (P < 0.01 or 0.05). CONCLUSIONS: Our present study indicated that EZF significantly inhibited the bone loss induced by OVX in mice by its regulation of estradiol combined with the nourishing effect of the uterus, and that it also attenuated bone resorption by decreasing the RANKL/OPG ratio so as to inhibit osteoclast maturation.

Network pharmacology strategy for revealing the pharmacological mechanism of pharmacokinetic target components of San-Ye-Tang-Zhi-Qing formula for the treatment of type 2 diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: San-Ye-Tang-Zhi-Qing formula (SYTZQ) is an effective prescription for the treatment of pre-diabetes disorders of glycolipid metabolism in type 2 diabetes mellitus (T2DM). It consists of five Chinese herbs including Mori Folium, Nelumbinis Folium, Crataegi Folium, Salviae Miltiorrhizae Radix et Rhizoma and Paeoniae Radix Rubra. AIM OF THE STUDY: This study was aimed to reveal the pharmacological mechanism of pharmacokinetic target components of SYTZQ for the treatment of T2DM. MATERIALS AND METHODS: A rapid, precise and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify simultaneously nuciferin, vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside, paeoniflorin and rosmarinic acid in rat plasma after oral administration of SYTZQ. The network pharmacology was used to analyze the effect of the compounds absorbed into the blood of SYTZQ on T2DM. The effects of paeoniflorin, nuciferine and rosmarinic acid on adipogenic differentiation were validated in vitro experiments. RESULTS: The separation was performed on an ACQUITY UHPLC HSS T3 column (2.1 mm × 100 mm, 1.7 µm) using acetonitrile and 0.1% (v/v) formic acid in water as the mobile phase in gradient elution. The calibration curves of five analytes showed good linearity (r ≥ 0.9991) with the lower limits of quantification (LLOQ) between 0.3 and 5.0 ng/mL. The recoveries and matrix effects of five analytes ranged from 81.1% to 113%. The RSDs of inter-day and intra-day precision were all within 13.7%. The validated method was successfully applied to the pharmacokinetic study of five ingredients after oral administration of SYTZQ to rat. 39 major targets and 22 candidate pathways of five compounds absorbed into the blood of rats after administration of SYTZQ were identified and successfully constructed a compound-target-disease-pathway network. It was confirmed that paeniforin, nuciferine and rosmarinic acid could decrease the adipogenicity differentiation in vitro experiments. CONCLUSIONS: The pharmacokinetic parameters indicated that the five components (nuciferin, vitexin-4″-O-glucoside, vitexin-2″-O-rhamnoside, paeoniflorin and rosmarinic acid) were absorbed and eliminated quickly in vivo. These five absorbed components were associated with 22 pathways, including insulin resistance, regulation of lipolysis in adipocytes, PI3k/AKT-, TNF-, cAMP- and cGMP-PKG-signaling pathway. Paeoniflorin, nuciferine and rosmarinic acid have the effect of inhibiting adipocyte differentiation. This study could provide more reference for quality control, and provide a firm basis for evaluating the clinical efficiency of SYTZQ.

Danhong injection attenuates isoproterenol-induced cardiac hypertrophy by regulating p38 and NF-κb pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI), derived from Rhizoma Salviae Miltiorrhizae (Salvia miltiorrhiza Bge., Labiatae, Danshen in Chinese) and Flos Carthami (Carthamus tinctorius L., Compositae, Salvia militiorrhiza Bunge), is an extensively-used Chinese material standardized clinical product for treatment of cardiovascular diseases. AIM OF THE STUDY: Cardiac hypertrophy (CH) is an adaptive response of cardiomyocytes. Long-lasting cardiac hypertrophy results in the loss of compensation by cardiomyocytes which could ultimately develop into heart failure. In the present study, we aimed to investigate the effect and exact mechanisms of DHI on isoproterenol (ISO)-induced CH. MATERIALS AND METHODS: H9c2 cells and male Wistar rats were stimulated by ISO in the present study to establish CH models in vitro and in vivo. CCk-8 assay, Western blot, real time-polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA) and Echocardiography were used in the present study. RESULTS: DHI significantly attenuated ISO-induced CH of H9c2 cells (p<0.01). DHI decreased ISO-induced atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) elevation both at the mRNA and protein levels (p<0.05 and p<0.01, respectively). Western blot showed that DHI down-regulated the phosphorylation of p38. Furthermore, we found that DHI inhibited the nuclear translocation and activation of NF-κb. Echocardiography from ISO-induced CH rats showed that DHI significantly decreased left ventricle (LV) mass, the thickness of the LV end-systolic posterior wall (LVPWs), and the LV end-diastolic posterior wall (LVPWd) elevated by ISO (p<0.01 and p<0.05, respectively). CONCLUSION: These data demonstrate that DHI might exert anti-cardiac hypertrophic effects by regulating p38 and NF-κb pathway.

Vasodilatory Effect of Wogonin on the Rat Aorta and Its Mechanism Study.

Wogonin, a natural flavonoid, is one of the bioactive compounds of the medicinal herb Eucommia ulmoides OLIV. widely used in southeastern Asia for treating hypertension. However, the molecular mechanisms for the therapeutic benefits remain largely unclear. The present study investigated the vasodilatory effect of wogonin and its possible mechanisms. The flavonoid (0.1-100 µM) caused concentration-dependent relaxations in endothelium-intact aortic rings precontracted with norepinephrine (NE, 1 µM) or potassium chloride (KCl, 60 mM). Preincubation with wogonin (10, 100 µM) for 20 min significantly inhibited the contractile responses to NE (0.1, 1, 10 µM) or KCl (7.5, 15, 30, 60 mM). Relaxant responses to wogonin were not inhibited by N(G)-nitro-L-arginine methylester (100 µM) or endothelial denudation. In a Ca(2+)-free Krebs' solution, wogonin not only blocked Ca(2+) influx-dependent vasoconstriction by either NE (1 µM) or KCl (100 mM), but also inhibited NE (1 µM)-induced tonic contraction, which is dependent on intracellular Ca(2+) release. Wogonin also suppressed the elevation of [Ca(2+)]i induced by KCl (60 mM) after exhausting the calcium store in sarcoplasmic and endoplasmic reticula with thapsigargin (1 µM) or by ATP (100 µM) in primary vascular smooth muscle cells. These findings suggest that wogonin-induced responses are mainly due to the inhibition of both intracellular Ca(2+) release and extracellular Ca(2+) influx.

Antidepressant-like effect of ethanol extract from Zuojin Pill, containing two herbal drugs of Rhizoma Coptidis and Fructus Evodiae, is explained by modulating the monoaminergic neurotransmitter system in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuojin Pill (ZJP), a traditional Chinese medicinal decoction, contains two herbal drugs: Coptis chinensis Franch. and Evodia rutaecarpa (Juss.) Benth. in the ratio of 6:1 (w/w). Previous pharmacological studies have shown that two herbs in ZJP have the antagonistic effects on catecholamine secretion in bovine adrenal medullary cells. Furthermore, the alkaloids from the two herbs in ZJP may provide a protective effect for depression in individuals with a low expressing 5-HTT allele by increasing receptor concentration in serotonergic neurons. However, antidepressant effect has not been reported before and has not been fully clarified. AIM OF THE STUDY: The present study aimed to investigate the antidepressant potential of ethanol extract from ZJP and its monoaminergic mechanism in mice. MATERIALS AND METHODS: Seven alkaloids were determined from the ethanol extract of ZJP using High Performance Liquid Chromatography (HPLC) with the gradient mobile phase. The ethanol extract from ZJP was used to evaluate the antidepressant potential in mice. Mouse models of depression including the tail suspension test (TST) and the forced swim test (FST) were used to evaluate the effects of the ethanol extract from ZJP. A possible mechanism was explored in the tests of antagonism of reserpine-induced ptosis and hypothermia, and 5-HTP induced head twitch response in mice. The contents of monoamine neurotransmitters including norepinephrine (NE), serotonin (5-hydroxytryptamine or 5-HT) in hippocampus of mice and NE, 5-HT, dopamine (DA) in striatum of mice were determined by HPLC system with Electrochemical Detector (ECD). RESULTS: The results showed that intragastric administration of the ethanol extract from ZJP (5, 10, 20mg/kg) or fluoxetine (7.5mg/kg) significantly reduced the duration of immobility in TST and FST. However, the effect was not dose-dependent. Ethanol extract from ZJP (5, 10, 20mg/kg) also increased the accumulative number of the 5-HTP-induced head twitch response in mice. The mice were treated with the ethanol extract from ZJP (5, 10, 20mg/kg) or fluoxetine (7.5mg/kg), which could antagonize reserpine-induced ptosis and hypothermia, moreover, both of them could elevate the contents of NE, 5-HT in hippocampus as well as NE, 5-HT, DA in striatum significantly. CONCLUSION: These results indicate that the ethanol extract from ZJP produced antidepressant-like effect and the possible mechanism, at least in part, is via the central monoaminergic neurotransmitter system and 5-HT plays a major role.

[Vasodilator effect of oroxylin A on thoracic aorta isolated from rats].

OBJECTIVE: To investigate the vasodilator effect and the endothelium-dependent mechanism of oroxylin A in thoracic aorta isolated from rats. METHODS: Thoracic aorta was isolated from Wistar rats. After pretreatment with norepinephrine or KCl, the effects of oroxylin A at different concentrations were detected on isolated vascular rings prepared from rats' thoracic aorta. The response of thoracic aortic ring was evaluated in the presence and absence of endothelium, and NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase. RESULTS: Oroxylin A (10 and 100 µmol/L) caused vasodilation on endothelium-intact aortic rings pretreated with norepinephrine (1 µmol/L) and KCl (60 mmol/L) compared with the control (P<0.05, P<0.01). The vasodilation function of 10 and 100 µmol/L oroxylin A on the endothelium-denuded aorta rings was significantly lower than that on the endothelium-intact aorta rings (P<0.05, P<0.01). L-NAME pretreatment significantly attenuated the effect of 100 µmol/L oroxylin A on endothelium-intact aorta rings (P<0.05, P<0.01). CONCLUSION: Oroxylin A can induce the relaxation of the aorta ring in endothelium-dependent manner. Nitric oxide may be involved in the endothelium-dependent effect of oroxylin A.