Orcinol gentiobioside inhibits RANKL-induced osteoclastogenesis by promoting apoptosis and suppressing autophagy via the JNK1 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoporosis (OP) is a metabolic disorder characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Curculigo orchioides Gaertn has a long history of application in traditional Chinese and Indian medicine for treating OP. Orcinol gentiobioside (OGB) is a principal active constituent derived from Curculigo orchioides Gaertn and has been shown to have anti-OP activity. However, the therapeutic efficacy and mechanism of OGB in modulating osteoclastic bone resorption remain undefined. AIM OF THE STUDY: To evaluate the effect of OGB on the formation, differentiation and function of osteoclasts derived from bone marrow macrophages (BMMs), and further elucidate the underlying action mechanism of OGB in OP. MATERIALS AND METHODS: Osteoclasts derived from BMMs were utilized to evaluate the effect of OGB on osteoclast formation, differentiation and bone resorption. Tartrate-resistant acid phosphatase (TRAP) staining and activity assays were conducted to denote the activity of osteoclasts. Osteoclast-related genes and proteins were determined by RT-PCR and Western blotting assays. The formation of the F-actin ring was observed by confocal laser microscopy, and bone resorption pits were observed by inverted microscopy. The target of OGB in osteoclasts was predicted by using molecular docking and further verified by Cellular Thermal Shift Assay (CETSA) and reversal effects of the target activator. The apoptosis of osteoclasts was analyzed by flow cytometry, and autophagic flux in osteoclasts was determined by confocal laser microscopy. RESULTS: OGB inhibited osteoclast formation and differentiation, osteoclast-related genes and proteins expression, F-actin ring formation, and bone resorption activity. Molecular docking and CETSA analysis demonstrated that OGB exhibited good affinity for c-Jun N-terminal Kinase 1 (JNK1). In addition, OGB induced apoptosis and inhibited autophagy in osteoclasts, and the JNK agonist anisomycin reversed the increase in apoptosis and inhibition of autophagy induced by OGB in osteoclasts. CONCLUSION: OGB inhibited osteoclastogenesis by promoting apoptosis and diminishing autophagy via JNK1 signaling.

[UPLC-Q-TOF-MS-based metabolomics reveals mechanism of Morinda officinalis iridoid glycosides in treating rheumatoid arthritis and bone loss].

This study aimed to investigate the therapeutic effects of Morinda officinalis iridoid glycosides(MOIG) on paw edema and bone loss of rheumatoid arthritis(RA) rats, and analyze its potential mechanism based on ultra-high performance liguid chromatography-guadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS) serum metabolomics. RA rats were established by injecting bovin type Ⅱ collagen. The collagen-induced arthritis(CIA) rats were administered drug by gavage for 8 weeks, the arthritic score were used to evaluate the severity of paw edem, serum bone metabolism biochemical parameters were measured by ELISA kits, Masson staining was used to observe the bone microstructure of the femur in CIA rats. UPLC-Q-TOF-MS was used to analyze the alteration of serum metabolite of CIA rats, principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were used to screen the potential biomarkers, KEGG database analysis were used to construct related metabolic pathways. The results demonstrated that the arthritic score, serum levels of IL-6 and parameters related with bone metabolism including OCN, CTX-Ⅰ, DPD and TRAP were significantly increased, and the ratio of OPG and RANKL was significantly decreased, the microstructure of bone tissue and cartilage were destructed in CIA rats, while MOIG treatments could significantly reduce arthritis score, mitigate the paw edema, reverse the changes of serum biochemical indicators related with bone metabolism, and improve the microstructure of bone tissue and cartilage of CIA rats. The non-targeted metabolomics results showed that 24 altered metabolites were identified in serum of CIA rats; compared with normal group, 13 significantly altered metabolites related to RA were identified in serum of CIA rats, mainly involving alanine, aspartate and glutamate metabolism; compared with CIA model group, MOIG treatment reversed the alteration of 15 differential metabolites, mainly involving into alanine, aspartate and glutamate metabolism, D-glutamine and D-glutamate metabolism, taurine and hypotaurine metabolism, valine, leucine and isoleucine biosynthesis. Therefore, MOIG significantly alleviated paw edema, improved the destruction of microstructure of bone and cartilage in CIA rats maybe through involving into the regulation of amino acid metabolism.

Effects of grafting on chemical constituents, toxicological properties, antithrombotic activity, and myocardial infarction protection of styrax secreted from the trunk of Liquidambar orientalis Mill.

Styrax, the balsam refined from the trunk of Liquidambar orientalis Mill. has a variety of applications in the perfumery and medical industry, especially for use in traditional Chinese medicine. However, the resources of styrax are in shortage due to being endangered of this plant. Grafting can improve the adaptability of plants to unfavorable environmental conditions. We tried to graft the L. orientalis Mill. on L. formosana Hance which was widely distributed in Jiangsu and Zhejiang provinces of China in an attempt to obtain styrax from grafted L. orientalis Mill. (grafted styrax, SG). Whether SG can become an alternative application of commercially available styrax (SC) need be further investigated. The components of SG were analyzed by GC-MS, and the results showed that the chromatograms of SG, SC, and styrax standard (SS) were consistent. The ration of 12 major chemical components based peak area in SG, SC, and SS were 93.95%, 94.24%, and 95.86% respectively. The assessment of toxicity, antithrombotic activity, and myocardial infarction protection of SG and SC was evaluated by using the zebrafish model, the results showed that SG and SC have the similar toxicological properties as evidenced by acute toxicity test, developmental toxicity and teratogenicity, and long-term toxicity test. Both SG and SC significantly decreased the thrombosis and increased blood flow velocity of zebrafish induced by adrenaline hydrochloride, inhibited myocardial apoptosis, myocardial infarction and myocardial inflammation in zebrafish induced by isoproterenol hydrochloride. Moreover, SG had an obvious improvement effect on cardiac output, while SC has no effect. Collectively, SG is similar to SC in chemical composition, toxicological properties, antithrombotic activity, and myocardial infarction protection effects, and may be used as a substitute for styrax to reduce the collection for wild L. orientalis Mill. and increase the available styrax resources.

A green, efficient and stable platform based on hyperbranched quaternized hydrothermal magnetic chitosan nanospheres integrated cytomembranes for screening drug candidates from natural products.

Compared with traditional tedious organic solvent-assisted separation process in natural medicinal chemistry, cytomembrane (CM) fishing technique became a more appealing and greener choice for screening bioactive components from natural products. However, its large-scale practical value was greatly weakened by the easy fall-off of CMs from magnetic supports, rooted in the instability of common Fe3O4 particles and their insufficient interaction with CMs. In this research, a new green biostable platform was developed for drug screening through the integration of hyperbranched quaternized hydrothermal magnetic carbon spheres (HQ-HMCSs) and CMs. The positive-charged HQ-HMCSs were constructed by chitosan-based hydrothermal carbonization onto Fe3O4 nanospheres and subsequent aqueous hyperbranching quaternization with 1,4-butanediol diglycidyl ether and methylamine. The strong interaction between HQ-HMCSs and CMs was formed via electrostatic attraction of HQ-HMCSs to negative-charged CMs and covalent linkage derived from the epoxy-amine addition reactions. The chemically stable HMCSs and its integration with CMs contributed to dramatically higher stability and recyclability of bionic nanocomposites. With the fishing of osteoblast CMs integrated HQ-HMCSs, two novel potential anti-osteoporosis compounds, narcissoside and beta-ionone, were discovered from Hippophae rhamnoides L. Enhanced osteoblast proliferation, alkaline phosphatase, and mineralization levels proved their positive osteogenesis effects. Preliminary pharmacological investigation demonstrated their potential action on membrane proteins of estrogen receptor alpha and insulin-like growth factor 1. Furthermore, beta-ionone showed apparent therapeutic effects on osteogenic lesions in zebrafish. These results provide a green, stable, cost-efficient, and reliable access to rapid discovery of drug leads, which verifiably benefits the design of nanocarbon-based biocomposites with increasingly advanced functionality.

Orcinol glucoside targeted p38 as an agonist to promote osteogenesis and protect glucocorticoid-induced osteoporosis.

BACKGROUND: Glucocorticoids (GC)-induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis, which leads to an increased risk of fracture in patients. The inhibition of the osteoblast effect is one of the main pathological characteristics of GIOP, but without effective drugs on treatment. PURPOSE: The aim of this study was to investigate the potential effects of orcinol glucoside (OG) on osteoblast cells and GIOP mice, as well as the mechanism of the underlying molecular target protein of OG both in vitro osteoblast cell and in vivo GIOP mice model. METHODS: GIOP mice were used to determine the effect of OG on bone density and bone formation. Then, a cellular thermal shift assay coupled with mass spectrometry (CETSA-MS) method was used to identify the target of OG. Surface plasmon resonance (SPR), enzyme activity assay, molecular docking, and molecular dynamics were used to detect the affinity, activity, and binding site between OG and its target, respectively. Finally, the anti-osteoporosis effect of OG through the target signal pathway was investigated in vitro osteoblast cell and in vivo GIOP mice model. RESULTS: OG treatment increased bone mineral density (BMD) in GIOP mice and effectively promoted osteoblast proliferation, osteogenic differentiation, and mineralization in vitro. The CETSA-MS result showed that the target of OG acting on the osteoblast is the p38 protein. SPR, molecular docking assay and enzyme activity assay showed that OG could direct bind to the p38 protein and is a p38 agonist. The cellular study found that OG could promote p38 phosphorylation and upregulate the proteins expression of its downstream osteogenic (Runx2, Osx, Collagen Ⅰ, Dlx5). Meanwhile, it could also inhibit the nuclear transport of GR by increasing the phosphorylation site at GR226 in osteoblast cell. In vivo GIOP mice experiment further confirmed that OG could prevent bone loss in the GIOP mice model through promoting p38 activity as well as its downstream proteins expression and activity. CONCLUSIONS: This study has established that OG could promote osteoblast activity and revise the bone loss in GIOP mice by direct binding to the p38 protein and is a p38 agonist to improve its downstream signaling, which has great potential in GIOP treatment for targeting p38. This is the first report to identify OG anti-osteoporosis targets using a label-free strategy (CETSA-MS).

Phylogeography of cultivated and wild ophiopogon japonicus based on chloroplast DNA: exploration of the origin and sustainable cultivation.

BACKGROUND: Ophiopogon japonicus, mainly planted in Sichuan (CMD) and Zhejiang (ZMD) province in China, has a lengthy cultivation history. During the long period of domestication, the genetic diversity of cultivated O. japonicus has substantially declined, which will affect the population continuity and evolutionary potential of this species. Therefore, it is necessary to clarify the phylogeography of cultivated O. japonicus to establish a theoretical basis for the utilization and conservation of the genetic resources of O. japonicus. RESULT: The genetic diversity and population structure of 266 O. japonicus individual plants from 23 sampling sites were analyzed based on 4 chloroplast DNA sequences (atpB-rbcL, rpl16, psbA-trnH and rpl20-5'rps12) to identify the effects of domestication on genetic diversity of cultivars and determine their geographic origins. The results showed that cultivated O. japonicus and wild O. japonicus had 4 and 15 haplotypes respectively. The genetic diversity of two cultivars (Hd = 0.35700, π = 0.06667) was much lower than that of the wild populations (Hd = 0.76200, π = 0.20378), and the level of genetic diversity in CMD (Hd = 0.01900, π = 0.00125) was lower than that in ZMD (Hd = 0.06900, π = 0.01096). There was significant difference in genetic differentiation between the cultivated and the wild (FST = 0.82044), especially between the two cultivars (FST = 0.98254). This species showed a pronounced phylogeographical structure (NST > GST, P < 0.05). The phylogenetic tree showed that the genetic difference between CMD and ZMD was not enough to distinguish the cultivars between the two producing areas by using O. amblyphyllus Wang et Dai as an outgroup. In addition, both CMD and ZMD have a closer relationship with wild populations in Sichuan than that in Zhejiang. The results of the TCS network and species distribution model suggested that the wild population TQ located in Sichuan province could serve as the ancestor of cultivated O. japonicus, which was supported by RASP analysis. CONCLUSION: These results suggest that cultivated O. japonicus has experienced dramatic loss of genetic diversity under anthropogenic influence. The genetic differentiation between CMD and ZMD is likely to be influenced by founder effect and strong artificial selection for plant traits. It appears that wild populations in Sichuan area are involved in the origin of not only CMD but also ZMD. In addition, we also raise some suggestions for planning scientific strategies for resource conservation of O. japonicus based on its genetic diversity and population structure.

Jintiange proteins promote osteogenesis and inhibit apoptosis of osteoblasts by enhancing autophagy via PI3K/AKT and ER stress pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Tiger bone, which had long been used in traditional Chinese medicine, had the action of removing wind and alleviating pain, strengthening the sinews and bones, and often used to treat bone impediment, and atrophic debility of bones in TCM clinical practice. As a substitute of natural bone tiger, artificial tiger bone Jintiange (JTG), has been approved by the State Food and Drug Administration of China for relief the symptom of osteoporosis, such as lumbago and back pain, lassitude in loin and legs, flaccidity and weakness legs, and walk with difficulty based on TCM theory. JTG has similar chemical profile to natural tiger bone, and contains mineral substance, peptides and proteins, and has been shown to protect bone loss in ovariectomized mice and exert the regulatory effects on osteoblast and osteoclast activities. But how the peptides and proteins in JTG modulate bone formation remains unclear. AIM: To investigate the stimulating effects of JTG proteins on osteogenesis and explore the possible underlying mechanisms. MATERIALS AND METHODS: JTG proteins were prepared from JTG Capsules by extracting calcium, phosphorus and other inorganic elements using SEP-PaktC18 desalting column. MC3T3-E1 cells were treated with JTG proteins to evaluate their effects and explore the underlying mechanisms. Osteoblast proliferation was detected by CCK-8 method. ALP activity was detected using a relevant assay kit, and bone mineralized nodules were stained with alizarin red-Tris-HCl solution. Cell apoptosis was analyzed by flow cytometry. Autophagy was observed by MDC staining, and autophagosomes were observed by TEM. Nuclear translocations of LC3 and CHOP were detected by immunofluorescence and observed under a laser confocal microscope. The expression of key proteins related to osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways was analyzed by Western Blot analysis. RESULTS: JTG proteins improved osteogenesis as evidenced by the alteration of proliferation, differentiation and mineralization of MC3T3-E1 osteoblasts, inhibited their apoptosis, and enhanced autophagosome formation and autophagy. They also regulated the expression of key proteins of PI3K/AKT and ER stress pathways. In addition, PI3K/AKT and ER stress pathway inhibitors could reverse the regulatory effects of JTG proteins on osteogenesis, apoptosis, autophagy and PI3K/AKT and ER stress pathways. CONCLUSION: JTG proteins increased the osteogenesis and inhibited osteoblast apoptosis by enhancing autophagy via PI3K/AKT and ER stress signaling pathways.

Establishment of Male and Female Eucommia Fingerprints by UPLC Combined with OPLS-DA Model and Its Application.

Eucommia ulmoides Oliver is a dioecious plant, which plays an important role in traditional Chinese medicine. However, there has not yet been any research on male and female E. ulmoides. The UPLC fingerprints and OPLS-DA approach were able to quickly and easily identify and quantify E. ulmoides and differentiate between the male and female fingerprints. In this study, we optimized the UPLC conditions and analyzed them to investigate fingerprints of twenty-four extracts of Eucommiae Cortex (EC) and twenty-four extracts of Eucommiae Folium (EF) under optimal conditions. It was demonstrated that thirteen and twelve substances were possible chemical markers for EC and EF male and female discrimination and that the level of these markers - chlorogenic acid and protocatechuic acid - was many times higher in male than in female. This approach offered a reference for quality control and precise treatment of male and female E. ulmoides in the clinic.

Extraction, characterization, and anti-nonalcoholic steatohepatitis activity of a (1,3) (1,6)-ß-D-glucan from the Polyporus umbellatus (Pers.) Fries.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by inflammation and hepatic steatosis that may coincide with fibrotic activity. To date, no pharmacological agents have been approved for NASH treatment. Here, a homogeneous (1,3),(1,6)-ß-D-glucan (PUP-W-1, Mw: 41.07 kDa) was successfully purified from Polyporus umbellatus (Pers.) Fries sclerotia and characterized. The analysis showed that the PUP-W-1 backbone consisted of a repeating chain of eight →3)-ß-D-Glcp-(1 â†’ units, with branched chains of four ß-D-Glcp residues, joined by repeating 1,6-linkage units at the O-6 position of the backbone. The pharmacological effects of PUP-W-1 treatment in the context of NASH pathogenesis were explored using a methionine choline-deficient (MCD) diet-induced murine steatohepatitis model. The MCD model mice exhibited pronounced steatohepatitis, inflammatory activity, steatosis, stellate cell activation, and mild fibrotic activity. Treatment of the mice for three weeks with PUP-W-1 prevented the development of NASH due to the suppression of inflammation, lipid accumulation, and fibrosis. As suggested by these findings, PUP-W-1 may hold promise as a natural drug candidate or precursor for the treatment of NASH.

Toxicological effects of fipronil on laying hens and its residue elimination in eggs.

Eighty 24-week-old laying hens were divided into eight groups, seven given a single oral dose per chicken with 7 dosing levels from 13.6 to 137 mg/kg body weight (bw) and one serving as sham control. The hens were observed for 28 days for clinical abnormalities, egg yield, and body weight. Egg samples from groups of low-to-medium doses were analyzed for residues of fipronil and its metabolites by LC-MS/MS. Blood and organ samples from hens of the group receiving 63.3 mg/kg bw were collected for hematochemical and histopathological analysis. We found that the median lethal dose (LD50) of fipronil was 74 mg/kg bw for laying hens. No death occurred, and there were no obvious changes in body weight and egg production in the hens receiving doses at or below 20 mg/kg bw. In the hens that survived exposure to the dose at 63.3 mg/kg bw, there was significant reduction in body weight and egg yield; histopathological changes in the liver and kidney; and increased levels of creatine, urea, glutamate oxaloacetate transferase, and glutamate pyruvic transaminase. Fipronil-sulfone was the residual marker in eggs with significantly higher concentrations and longer withdrawal periods than its maternal compound. We conclude that fipronil is efficiently transformed into fipronil-sulfone in the body with subsequent excretion into eggs. More attention should be paid to the potential food safety risk of fipronil-sulfone because of its persistence in eggs.

[Research progress on anti-inflammatory effects of plant-derived cannabinoid type 2 receptor modulators].

Excessive and persistent inflammatory responses are a potential pathological condition that can lead to diseases of various systems, including nervous, respiratory, digestive, circulatory, and endocrine systems. Cannabinoid type 2 receptor(CB2R) belongs to the G protein-coupled receptor family and is widely distributed in immune cells, peripheral tissues, and the central nervous system. It plays a role in inflammatory responses under various pathological conditions. The down-regulation of CB2R activity is an important marker of inflammation and and CB2R modulators have been shown to have anti-inflammatory effects. This study explored the relationship between CB2R and inflammatory responses, delved into its regulatory mechanisms in inflammatory diseases, and summarized the research progress on CB2R modulators from plants other than cannabis, including plant extracts and monomeric compounds, in exerting anti-inflammatory effects. The aim is to provide new insights into the prevention and treatment of inflammatory diseases.

Jin-Tian-Ge ameliorates ovariectomy-induced bone loss in rats and modulates osteoblastogenesis and osteoclastogenesis in vitro.

BACKGROUND: Tiger bone, which had been one of the most famous traditional Chinese medicine for 2000 years, was originate from the skeleton of Panthera tigris L., and had the actions of anti-inflammatory, analgesic, immune-regulatory and promoting healing of bone fracture, and was used for the treatment of osteoporosis and rheumatoid arthritis. Jin-Tian-Ge (JTG), the artificial tiger bone powder, were prepared from skeletons of several farmed animals to substitute the natural tiger bone, and has been used for the treatment of osteoporosis in clinical practice. However, the characteristic and mechanism of action of JTG for the therapy of osteoporosis need to be further evidenced by using modern pharmacological methods. The aim of this work is to investigate the bone-protective effects of JTG, and explore the possible underlying mechanism. METHODS: Ovariectomy (OVX) rats were orally administrated JTG or estradiol valerate (EV) for 12 weeks. We investigated the pharmacodynamic effects of JTG on anti-bone loss in OVX rats, and also investigated the role of JTG in promoting osteogenesis and inhibiting osteoclast differentiation. RESULTS: JTG increased the bone mineral density (BMD), improved the bone microarchitecture and biomechanical properties in ovariectomized rast, whereas reversed the bone high turnover in OVX rats as evidenced by serum biochemical markers in OVX rats. JTG increased osteogenic differentiation of BMSCs in vitro, and up-regulated the expression of the key proteins of BMP and Wnt/ß-catenin pathways. JTG also inhibited the osteoclastogenesis of BMM as evidenced by the alteration of the TRAP activity, F-actin construction and the expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), c-Fos, Cathepsin K (Ctsk) and matrix metallopeptidase 9 (MMP9) of OCs induced with RANKL and LPS, reduced the expression and phosphorylation of NF-κB in OCs. CONCLUSIONS: JTG prevented bone loss in OVX rats and increased osteogenic differentiation of BMSCs through regulation of the BMP and Wnt/ß-catenin pathway, inhibited osteoclastogenesis by suppressing the NF-κB pathway, suggesting that JTG had the potentials for prevention and treatment of osteoporosis by modulating formation and differentiation of osteoblast and osteoclast.

Monotropein Protects against Inflammatory Bone Loss and Suppresses Osteoclast Formation and Bone Resorption by Inhibiting NFATc1 via NF-κB and Akt/GSK-3ß Pathway.

Monotropein (Mon) is a kind of iridoid glycoside plant secondary metabolite primarily present in some edible and medicinal plants. The aim of this study was to investigate the effect of Mon on lipopolysaccharide (LPS)-induced inflammatory bone loss in mice and osteoclasts (OCs) derived from bone marrow-derived macrophages (BMMs), and explore the mechanisms underlying the effect of Mon on LPS-induced osteoclastogenesis. It was found that Mon markedly attenuated deterioration of the bone micro-architecture, enhanced tissue mineral content (TMC) and bone volume/total volume (BV/TV), reduced structure model index (SMI) and trabecular separation/spacing (Tb.Sp) in the bone tissue and decreased the activities of tartrate resistant acid phosphatase-5b (TRACP-5b), receptor activator NF-κB (RANK), and receptor activator NF-κB ligand (RANKL) as well as the serum levels of interleukin 6 (IL-6) and interleukin 1ß (IL-1ß) in LPS-treated mice. In addition, Mon treatment reduced the number of TRAP positive OCs in the bone tissue of LPS-treated mice and also exerted a stronger inhibitory effect on formation, differentiation, and F-actin ring construction of OCs derived from BMMs. Mon significantly inhibited the expression of the nuclear factor of activated T-cells c1 (NFATc1) and the immediate early gene (C-Fos) and nuclear translocation of NFATc1 in LPS-treated OCs, thereby inhibiting the expression of matrix metalloproteinase-9 (MMP-9), cathepsin K (CtsK), and TRAP. Mon significantly inhibited the expression of TRAF6, phosphorylation of P65, and degradation of IKBα, thus inhibiting the activation of NF-κB pathway in LPS-induced inflammatory mice and OCs derived from BMMs, and also inhibited LPS-induced phosphorylation of protein kinase B (Akt) and Glycogen synthase kinase 3ß (GSK-3ß) in OCs derived from BMMs. In conclusion, these results suggested that Mon could effectively inhibit osteoclastogenesis both in vitro and in vivo and therefore may prove to be potential option for prevention and treatment of osteoclastic bone resorption-related diseases.

Application progress of immobilized biomembrane in the discovery of active compounds of natural products.

Natural products (NP) are an important source of bioactive compounds. Considering their complex matrix effects, the development of suitable methodologies for the quick identification and analysis of active substances in NPs played a significant role in controlling their quality and discovering new drugs. In recent years, the technology of immobilized biomembrane has attracted increasing attention, due to its advantages such as multitarget efficiency, accuracy, and/or time-saving compared with traditional activity-guided separation and ligand fishing methods. This article provides a systematic review of the latest advances in screening technologies based on biomembrane in the field of NPs. It includes detailed discussions on these technologies, including cell membrane chromatography, artificial membrane chromatography, cell membrane fishing, living cell fishing methods, and their applications in screening various active molecules from NPs. Their limitations and future development prospects are further discussed.

Orcinol Glucoside Improves Senile Osteoporosis through Attenuating Oxidative Stress and Autophagy of Osteoclast via Activating Nrf2/Keap1 and mTOR Signaling Pathway.

Oxidative stress and autophagy play essential roles in the development of senile osteoporosis which is characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Orcinol glucoside (OG), a phenolic glycoside isolated from Curculigo orchioides Gaertn, possesses antiosteoporotic properties. This study examined the protective effects of OG on bone loss in SAMP6 mice and explored the underlying mechanisms. The osteoporotic SAMP6 mice were treated with OG oral administration. RAW264.7 cells were induced to differentiate into osteoclast by RANKL and H2O2 in vitro and received OG treatment. The results demonstrated that OG attenuated bone loss in SAMP6 mice and inhibited the formation and bone resorption activities of osteoclast and reduced levels of oxidative stress in bone tissue of SAMP6 mice and osteoclast. Furthermore, OG activated Nrf2/Keap1 signaling pathway and enhanced the phosphorylation of mTOR and p70S6K which are consequently suppressing autophagy. Of note, the effect of OG on Nrf2/Keap1 signaling was neutralized by the mTOR inhibitor rapamycin. Meanwhile, the inhibitory effect of OG on autophagy was reversed by the Nrf2 inhibitor ML385.Conclusively, OG attenuated bone loss by inhibiting formation, differentiation, and bone resorption activities of osteoclast. Regulation of Nrf2/Keap1 and mTOR signals is a possible mechanism by which OG suppressed oxidative and autophagy of osteoclasts. Thus, OG prevented senile osteoporosis through attenuating oxidative stress and autophagy of osteoclast via activating Nrf2/Keap1 and mTOR signaling pathway.

MSC Transplantation Attenuates Inflammation, Prevents Endothelial Damage and Enhances the Angiogenic Potency of Endogenous MSCs in a Model of Pulmonary Arterial Hypertension.

Purpose: Pulmonary arterial hypertension (PAH) is a progressive and fatal pulmonary vascular disease initiated by endothelial dysfunction. Mesenchymal stromal cells (MSCs) have been shown to ameliorate PAH in various rodent models; however, these models do not recapitulate all the histopathological alterations observed in human PAH. Broiler chickens (Gallus gallus) can develop PAH spontaneously with neointimal and plexogenic arteriopathy strikingly similar to that in human patients. Herein, we examined the protective effects of MSC transplantation on the development of PAH in this avian model. Methods: Mixed-sex broilers at 15 d of age were received 2×106 MSCs or PBS intravenously. One day later, birds were exposed to cool temperature with excessive salt in their drinking water to induce PAH. Cumulative morbidity from PAH and right-to-left ventricle ratio were recorded. Lung histologic features were evaluated for the presence of endothelial damage, endothelial proliferation and plexiform lesions. Expression of proinflammatory mediators and angiogenic factors in the lung was detected. Matrigel tube formation assay was performed to determine the angiogenic potential of endogenous MSCs. Results: MSC administration reduced cumulative PAH morbidity and attenuated endothelial damage, plexiform lesions and production of inflammatory mediators in the lungs. No significant difference in the expression of paracrine angiogenic factors including VEGF-A and TGF-ß was determined between groups, suggesting that they are not essential for the beneficial effect of MSC transplantation. Interestingly, the endogenous MSCs from birds receiving MSC transplantation demonstrated endothelial differentiatial capacity in vitro whereas those from the mock birds did not. Conclusion: Our results support the therapeutic use of MSC transplantation for PAH treatment and suggest that exogenous MSCs produce beneficial effects through modulating inflammation and endogenous MSC-mediated vascular repair.

Streptomyces griseicoloratus sp. nov., isolated from soil in cotton fields in Xinjiang, China.

A novel bacterium of the genus Streptomyces, designated TRM S81-3T, was isolated from soil in cotton fields of Xinjiang, China. Comparative 16S rRNA gene sequence analysis indicated that strain TRM S81-3T is most closely related to Streptomyces viridiviolaceus NBRC 13359T (98.9% sequence similarity); however, the average nucleotide identity (ANI) between strains TRM S81-3T and S. viridiviolaceus NBRC 13359T is relatively low (91.6%). Strain TRM S81-3T possesses LL-diaminopimelic acid as the diagnostic cell-wall diamino acid, MK-9(H4), MK-9(H6), and MK-9(H10) as the major menaquinones, and polar lipids including diphosphatidylglycerol (DPG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylmethyl ethanolamine (PME), phosphotidylinositolone (PI), phospholipid of unknown structure containing glucosamine (NPG), and two unidentified phospholipids (PLs).The major fatty acids are iso-C16:0, anteiso-C15:0, anteiso-C17:1 ω9c, anteiso-C17:0, iso-C15:0, and C14:0. The genomic DNA G + C content is 72.1%. Based on the evidence from this polyphasic study, strain TRM S81-3T represents a novel species of Streptomyces, for which the name Streptomyces grisecoloratus is proposed. The type strain is TRM S81-3T (= CCTCC AA 2020002T = LMG 31942T).

Chinese herbal formula Tongxie Yaofang for diarrhea-predominant irritable bowel syndrome: study protocol for a randomized, multiple-blind, placebo-controlled trial.

INTRODUCTION: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a bowel disease with a high incidence. It significantly reduces the quality of life of patients and affects the patient's daily activities and mental health. No specific therapeutic medications for IBS-D have been found. Published clinical trials suggest that Chinese herbal formula Tongxie Yaofang (TXYF) for IBS-D may be effective. However, high-quality clinical evidence supporting its use in IBS-D is still lacking. This trial aims to evaluate the therapeutic effects and safety of TXYF for IBS-D in adults. METHODS/DESIGN: A randomized, multiple-blind, placebo-controlled trial will be conducted. It will consist of an 8-week intervention followed by a 3-month follow-up period. The target sample size is 96 IBS-D patients aged 18 to 65 years. The eligible participants will be randomly allocated to either TXYF or placebo group in a ratio of 1:1. Participants in the experimental group will take TXYF granules, while participants in the control group will be given TXYF placebo granules. The primary outcome will be the degree of IBS symptom severity measured using the scale of IBS symptom severity score. The secondary outcomes include: (a) stool frequency, and (b) stool consistency measured using the Bristol stool scale, (c) quality of life measured using the scale of IBS-quality of life, (d) anxiety measured using the self-rating anxiety scale, (e) depression measured by the self-rating depression scale, and (f) the safety of using TXYF and placebo. Safety monitoring and assessment will be undertaken throughout treatment. DISCUSSION: Chinese herbal formula TXYF consists of four Chinese herbs: Atractylodes macrocephala Koidz., Paeonia lactiflora Pall ., Citrus × aurantium L ., and Radix saposhnikoviae. It has been used for diarrhea for hundreds of years and may have a potential benefit in treating adults with IBS-D, but due to lack of high-quality evidence, we designed a randomized, multiple-blind, placebo-controlled trial to evaluate its therapeutic effects and safety. This trial will provide important data to guide the clinical practice of TXYF for the treatment of IBS-D in adults. TRIAL REGISTRATION: ISRCTN registry ISRCTN12453166 . Registered on 23 March 2021.

Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects.

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects-including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism-without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors-ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.

Sustainable hydrophilic ultrasmall carbonaceous spheres modified by click reaction for high-performance polymeric ion chromatographic stationary phase.

Novel poly(ethylvinylbenzene-divinylbenzene) (EVB-DVB) agglomerated with ultrasmall carbonaceous spheres (UCSs) anion-exchange packings for ion chromatography (IC) were constructed. Hydrophilic UCSs with mean sizes of 62-98 nm were synthesized in quantity by the polydiallyl dimethyl ammonium chloride aided hydrothermal carbonization of fructose. The green strategy based on the thiol-ene click reaction with cysteamine in aqueous system was first designed for the hyperbranched polyquaternary amine (HPA) grafting of UCSs with negligible damage on their monodispersity. The HPA modified UCSs were evenly distributed on sulfonated EVB-DVB substrate to form one uniform layer of functional nanospheres without observable coagulum. Seven typical anions (F-, Cl-, NO2-, Br-, NO3-, SO42- and PO43-) were baseline separated on constructed packing in 5 min with high efficiencies in the range of 44,800-71,100 plates m - 1. The rapid separation of polarizable anions, small organic acids and saccharides could be also accomplished under isocratic elution with competitive peak symmetry and efficiency. Good reproducibility was demonstrated by consecutive injection. Thiosulfate in water reducer was further detected on prepared packing in 4 min with detection limit of 0.04 mg L - 1 (S/N = 3) and good repeatability.