Mechanism investigation of Duhuo Jisheng pill against rheumatoid arthritis based on a strategy for the integration of network pharmacology, molecular docking and in vivo experimental verification.

CONTEXT: Duhuo Jisheng pill (DHJS) is a classic traditional Chinese medicine (TCM) formula for rheumatoid arthritis (RA). The effective components and therapeutic mechanisms of DHJS for treating RA are still unclear. OBJECTIVE: To explore the potential mechanism of DHJS against RA by means of network pharmacology and experimental verification. MATERIALS AND METHODS: A network pharmacology and molecular docking analysis based on phytochemistry was used to elucidate the mechanism of DHJS against RA. The targets of DHJS anti-RA active ingredient were obtained by searching TCMSP, ETCM and TCMSID. The RA model induced by collagen was established in Wistar rats. The rats in the DHJS group were administered doses of 0.5, 1.0 and 2.0 g/kg for a period of 10 d. The expression of targets was measured with Western blot. RESULTS: Network pharmacology analysis showed that the anti-RA effect of DHJS was mediated by targets involved in immunity, inflammation and apoptosis, as well as PI3K-Akt and NF-κB signalling pathways. Of 2.0 g/kg DHJS significantly alleviated the ankle inflammation (IL-6: 62.73 ± 8.39 pg/mL, IL-1ß: 50.49 ± 11.47 pg/mL, TNF-α: 16.88 ± 3.05 pg/mL, IL-17A: 12.55 ± 1.87 pg/mL, IL-10: 16.24 ± 3.00 pg/mL), comparing with the model group (IL-6: 92.02 ± 13.25 pg/mL, IL-1ß: 71.85 ± 4.12 pg/mL, TNF-α: 25.64 ± 3.69 pg/mL, IL-17A: 22.14 ± 4.56 pg/mL, IL-10: 9.51 ± 3.03 pg/mL) (p < 0.05). Moreover, the protein expression of p-PI3K, p-AKT and p-p65 significantly decreased after DHJS administration. CONCLUSIONS: DHJS could alleviate the collagen-induced arthritis (CIA) by the PI3K/AKT/NF-κB signalling pathway.

The Antitumor Constituents from Hedyotis Diffusa Willd.

As a TCM, Hedyotis diffusa Willd. has been using to treat malignant tumors, and many studies also showed that the extracts from Hedyotis diffusa Willd. possessed evident antitumor activities. Therefore, we carried out chemical study on Hedyotis diffusa Willd. and investigated the cytotoxicity of the obtained compounds on a panel of eight tumor cell lines. As a result, four new compounds were isolated from Hedyotis diffusa Willd., including three iridoid glycosides of Shecaoiridoidside A-C (1-3) and a cerebroside of shecaocerenoside A (4). Also, six known iridoid compounds (5-10) were also obtained. The cytotoxicity of all compounds against human tumor cell lines of HL-60, HeLa, HCT15, A459, HepG2, PC-3, CNE-2, and BCG-823 were also evaluated in vitro. New compound 3 exhibited evident cytotoxicity to all tumor cell lines except the Hela, and the IC50 values are from 9.6 µM to 62.2 µM, while new compound 4 showed moderate cytotoxicity to all the cell lines, and the IC50 values are from 33.6 µM to 89.3 µM. By contrast, new compound 1 and known compound 9 showed moderate cytotoxicity to HCT15, A459, and HepG2 selectively. Known compound 7 also exhibited moderate cytotoxicity to HCT15 and A459 selectively.

Formulation and in vitro characterization of a novel solid lipid-based drug delivery system.

The liquid self-emulsifying drug delivery system (L-SEDDS), commonly used to deliver effective but poorly water-soluble oleanolic acid (OA), has many limitations such as high manufacturing costs, few choices of dosage forms, risk of leakage from hard gelatin capsules, low stability, limited portability, incompatibility with capsule materials, and relatively restricted storage conditions. Thus the main purpose of our study was to develop a promising solid lipid-based drug delivery system (S-SEDDS) for OA. The S-SEDDS, prepared from wet granulation with an optimized L-SEDDS formulation and mannitol, was characterized by particle size analysis, scanning electron microscopy, differential scanning calorimetry, and X-ray powder diffraction. Finally, the solubility of the OA-loaded S-SEDDS was compared with that of OA powder in the dissolution assay. Our new S-SEDDS for OA was developed from the optimum L-SEDDS with ethyl oleate (oil phase), Labrasol (surfactant), and Transcutol P (cosurfactant) at a volume ratio of 15:71:14 with 1.5% w/v OA and mannitol. The dissolution of OA was improved by 60% compared with that of the pure OA powder. All the problems associated with the L-SEDDS were resolved. The methodologies we developed for OA delivery could also be utilized for the delivery of other drugs with the S-SEDDS.

Percent Recovery Index Predicts Poor Asthma Control and Exacerbation in Adults.

Background: Previous studies indicate that the percent recovery index (PRI: the percentage increase from the maximally reduced FEV1 after bronchodilator inhalation), one of the indexes of methacholine bronchial provocation, may predict acute asthma exacerbations in childhood and elderly asthmatics. It is known that childhood (<12) and elder (>60) asthmatics may be different to adult patients in many aspect including prognosis. However, in adults, a research for predicting value of PRI to exacerbation is still absence. Besides exacerbation, predicting value of PRI to poor asthma control is also unknown. We try to detect whether PRI can predict poor asthma control and exacerbation in adults in this research. Meanwhile, we try to detect whether treatment can influence PRI. Methods: In 61 adults with asthma, baseline PRI was measured during enrollment. And then baseline PRI was evaluated as a predictor of exacerbation or poor asthma control at an upcoming 3-month follow-up. The covariates included age, sex, BMI, previous exacerbation, smoking status and baseline lung function. After treatment for 3 months, PRI was measured again and compared with baseline PRI. Results: After the 3-month follow-up, we found that baseline PRI was significantly related to asthma exacerbation (P = 0.023), poor asthma control (ACT at 3 months, P = 0.014), decreased quality of life (decrease of MiniAQLQ, P = 0.010) and cumulative number of EDHO at 3 months (P = 0.039). Meanwhile, no significant correlation was observed between baseline PRI and inflammation factors (FENO, CaNO, and EOS). Finally, PRI was dramatically reduced after standard treatment for 3 months. Conclusion: PRI is efficient in the prediction of poor asthma control and exacerbation in adults. The predictive value of PRI may rely on the inherent property of asthmatic airway smooth muscle (ASM) independent of inflammation factors. Effective treatment can alleviate PRI dramatically and that indicate PRI may also be valuable in evaluation of curative effect.

The association between vitamin D receptor gene polymorphisms and asthma: a systematic review and meta-analysis.

BACKGROUND: Numerous studies have reported on the genetic factors related to asthma. In recent years, the vitamin D receptor (VDR) has been identified as one of the asthma susceptibility genes that is closely associated with the pathogenesis of asthma. METHODS: Randomized controlled trials (RCTs) related to asthma and the VDR were identified from the Chinese and English databases. The following keywords were used as search terms: "asthma", "vitamin D receptor", "VDR", "polymorphism", and "mutation". Meta-analysis was performed using RevMan 5.3 and Stata 13 software provided by the Cochrane system. RESULTS: A total of 7 RCTs were included in this meta-analysis, 6 of which described the correct random allocation methods, 6 described the allocation plan in detail, and 4 used the blinding method. The frequency of the CC + CA dominant genotype at the Apa I locus and the GG + GA genotype frequency at the Bsm I locus of the VDR gene were significantly higher in asthmatic patients compared to control healthy patients [odds ratio (OR) =0.81, 95% confidence interval (CI): 0.68 to 0.98, P=0.03<0.05; and OR =2.05, 95% CI: 1.23 to 3.41, P=0.006<0.05, respectively]. There were no significant differences between the CC, CT, and TT genotype frequencies at the Fok I site of the VDR gene in the experimental group and the CC, CT, TT genotype frequencies at the Taq I site and the control group (P>0.05). There was no significant difference between the genotype frequencies. DISCUSSION: Meta-analysis confirmed that VDR gene polymorphisms are closely related to the onset of asthma, and the gene expression of the Fok I, Bsm I, Apa I, and Taq I loci directly affects the incidence of asthma.

[Mating system and causes of low fructification rate by self-pollination of Codonopsis pilosula].

OBJECTIVE: To study four kinds of germplasm resources of Codonopsis pilosula and provide the basal mating systems data for the breeding and cultivation of C. pilosula. METHOD: 0.5% TTC (2,3,5-triphenyl tetrazolium chloride) solution was used for the pollen viability test and benzidineand-H2O2 [1% benzidine in 60% ethanol,hydrogen peroxide (3%), and water, 4:11:22] was used for estimation of the stigma receptivity. The mating systems were tested by out crossing index (OC1), pollen-ovule ratio (P/O) and pollination by bagged and emasculated in the field. RESULT: The pollen viability of C. pilosula reached highly about 80% when the pollen staying in the anther, 2-3 days before the petals opening. The anther began scattering pollen before the day of the petals opening, the pollen viability was the highest about 95%, the pollen stick thickly aroud the stigma and quickliy lost in the next day. The stigma life-span was about 4-5 days, the optimal time for pollination was the first day of the petals opening, when the stigma was highly sticky and yellow. The value of out crossing index (OC1) was 4, pollen-ovule ratio was between 104.84-185.75. The natural fructification rate of cross-pollination by emasculated-treatment was 25.6% 42.4%. The fructification rate and compatible index of self-pollination by bagged- treatment were about 3.3%-6.7% and 3.0-21.8. CONCLUSION: The mating system of C. pilosula is mixed with self-pollination and cross-pollination, prone to cross-pollination. The compatibility of self-pollination is high. The difference of maturing period of pistil and stamen and the lack of polen amount cause low fructification rate of self-pollination.

The role of JAK/STAT signaling pathway and its inhibitors in diseases.

The JAK/STAT signaling pathway is an universally expressed intracellular signal transduction pathway and involved in many crucial biological processes, including cell proliferation, differentiation, apoptosis, and immune regulation. It provides a direct mechanism for extracellular factors-regulated gene expression. Current researches on this pathway have been focusing on the inflammatory and neoplastic diseases and related drug. The mechanism of JAK/STAT signaling is relatively simple. However, the biological consequences of the pathway are complicated due to its crosstalk with other signaling pathways. In addition, there is increasing evidence indicates that the persistent activation of JAK/STAT signaling pathway is closely related to many immune and inflammatory diseases, yet the specific mechanism remains unclear. Therefore, it is necessary to study the detailed mechanisms of JAK/STAT signaling in disease formation to provide critical reference for clinical treatments of the diseases. In this review, we focus on the structure of JAKs and STATs, the JAK/STAT signaling pathway and its negative regulators, the associated diseases, and the JAK inhibitors for the clinical therapy.

'Prodrug-Like' Acetylmannosamine Modified Liposomes Loaded With Arsenic Trioxide for the Treatment of Orthotopic Glioma in Mice.

Glioma is one of the fatal intracranial cancers that is a huge challenge to decrease the death rate currently. The deep penetration and high accumulation of therapeutic inorganic ions into the tumor site are extremely impeded due to the existence of physiological barriers, which limits to widen the indication of some drugs such as arsenic trioxide. The previous data have confirmed that the mannose substrate (MAN) without acetyl groups facilitates vesicles to go into the brain. Given that deacetylation of Ac4MAN groups on the surface of liposomes under the enzyme incubation occurred, namely 'prodrug-like' features of vesicles, the liposomes could more easily penetrate the BBB, target the glioma site, release arsenic trioxide, and inhibit the growth of glioma cells in the brain. Besides, the possibility of Ac4MAN binding to Gluts could be reduced due to the steric hindrance of acetyl groups, decreasing the off-target effects of vesicles. Here, we developed 'prodrug-like' arsenic trioxide (As2O3, ATO)-loaded liposomes inserted with distearoyl phospho-ethanolamine-polyethylene glycol-1000-p-carboxylpheny-α-d-acetylmannosamine (DSPE-PEG-1000-Ac4MAN), which was named Ac4MAN-ATO-LIP. Cytotoxic experiments of liposomes indicated that the toxicity of Ac4MAN-ATO-LIP was lower than that of free ATO but stronger than that of ATO-LIP (without insertion of DSPE-PEG-1000-Ac4MAN). The uptake of vesicles by U87 glioma cells displayed that the cellular uptake of Ac4MAN-Rho-LIP (labeled by rhodamine) was remarkably improved, compared with Rho-LIP. The in vivo biodistribution results showed the superiority of Ac4MAN-Rho-LIP in enhanced intracranial accumulation. Furthermore, the treatment of orthotopic glioma in Balb/c nude mice with Ac4MAN-ATO-LIP elongated the survival time of the animals than that with physiological saline, free ATO, or ATO-LIP, respectively. All the results suggested that the Ac4MAN-ATO-LIP had stronger anti-glioma effects as well as lower toxicities, and may be a promising approach for the treatment of brain cancer.

Iridoids and sfingolipids from Hedyotis diffusa.

Seven new compounds were isolated from the aerial part of Hedyotis diffusa, including three iridoid glycosides, hedyoiridoidside A - C (1-3), two cerebrosides, hedyocerenoside F (4) and G (5), and two new ceramides, hedyoceramide A (6) and B (7). And six known iridoid glycosides (8-13) were also obtained. Their structures were established by their physico-chemical constants and spectroscopic analysis. The cytotoxicity of all compounds against tumor cell lines of human cervical cancer HeLa, human leukemia HL-60, human lung cancer A459, human hepatoma HepG2, human gastric gland carcinoma BCG-823, human nasopharyngeal cancer CNE-2, human colon cancer HCT15, and human prostate cancer PC-3 were also evaluated in vitro. As a result, new compound 1 exhibited evident cytotoxicity to all tumor cell lines, and the IC50 values are from 9.5µM to 28.2µM, while new compound 2 exhibited evident cytotoxicity to Hela, HL-60, A459, HepG2, BGC-823, CNE-2, and HCT15, and the IC50 values are from 15.8µM to 26.2µM. Known compound 11 also exhibited evident cytotoxicity to HL-60, A459, HepG2, BGC-823, CNE-2, and HCT15, and the IC50 values are from 16.5µM to 40.4µM. New compounds 4-7 and known compounds 12 and 13 showed moderate cytotoxicity to some tumor cell lines.

NaF-loaded core-shell PAN-PMMA nanofibers as reinforcements for Bis-GMA/TEGDMA restorative resins.

A kind of core-shell nanofibers containing sodium fluoride (NaF) was produced and used as reinforcing materials for dimethacrylate-based dental restorative resins in this study. The core-shell nanofibers were prepared by coaxial-electrospinning with polyacrylonitrile (PAN) and poly(methyl methacrylate) (PMMA) solutions as core and shell fluids, respectively. The produced PAN-PMMA nanofibers varied in fiber diameter and the thickness of PMMA shell depending on electrospinning parameters. NaF-loaded nanofibers were obtained by incorporating NaF nanocrystals into the core fluid at two loadings (0.8 or 1.0wt.%). Embedment of NaF nanocrystals into the PAN core did not damage the core-shell structure. The addition of PAN-PMMA nanofibers into Bis-GMA/TEGDMA clearly showed the reinforcement due to the good interfacial adhesion between fibers and resin. The flexural strength (Fs) and flexural modulus (Ey) of the composites decreased slightly as the thickness of PMMA shell increasing. Sustained fluoride releases with minor initial burst release were achieved from NaF-loaded core-shell nanofibers and the corresponding composites, which was quite different from the case of embedding NaF nanocrystals into the dental resin directly. The study demonstrated that NaF-loaded PAN-PMMA core-shell nanofibers were not only able to improve the mechanical properties of restorative resin, but also able to provide sustained fluoride release to help in preventing secondary caries.

Identification of active compounds from Caesalpinia sappan L. extracts suppressing IL-6 production in RAW 264.7 cells by PLS.

ETHNOPHARMACOLOGICAL RELEVANCE: Caesalpinia sappan L. is distributed in Southeast Asia and also used as herbal medicine for the treatment of various diseases such as burning sensations, leprosy, dysentery, osteoarthritis and rheumatoid arthritis (RA). The overproduction of IL-6 plays an important role in the prognosis of RA, but the active compounds from the extracts of Caesalpinia sappan L. suppressing IL-6 production remain unknown. AIMS OF THE STUDY: Identifying the main active compounds of Caesalpinia sappan L. extracts inhibiting the IL-6 production in LPS-stimulated RAW 264.7 cells by partial least squares (PLS). MATERIALS AND METHODS: Sixty-four samples with different proportions of compounds were prepared from Caesalpinia sappan L. by supercritical CO2 fluid extraction (SCFE) and refluxing. Each of 64 samples was applied to RAW 264.7 cells with LPS to evaluate whether IL-6 production by LPS is affected by addition of each sample. The IL-6 production in medium was determined by ELISA and the inhibitory activity of each sample was analyzed. In addition, the fingerprints of these 64 samples were also established by ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-MS). We used the PLS, a simplified method, to evaluate the results from IL-6 production and fingerprints. RESULTS: Each of 64 samples markedly suppressed LPS-induced IL-6 production in RAW cells. The fingerprints by UPLC-MS clearly revealed variations among 64 samples produced in different extract conditions. The PLS analysis with IL-6 production and fingerprints by UPLC-MS suggested that the peaks 71, 93, 150, 157, 168 have more influence on the inhibitory activity of Caesalpinia sappan L. extracts. The peaks 71, 93, 150 are likely representing sappanone A, protosappanin E and neoprotosappanin, respectively. The peaks 157 and 168 are still at large. CONCLUSION: This is the first report that sappanone A, protosappanin E, neoprotosappanin and two unidentified compounds can be considered as possible active compounds that might inhibit IL-6 production. Further studies are needed to confirm the effectiveness of these five compounds on IL-6 production and possible mechanism.

The Effects of Lactidyl/Glycolidyl Ratio and Molecular Weight of Poly(D,L -Lactide-co-Glycolide) on the Tetracycline Entrapment and Release Kinetics of Drug-Loaded Nanofibers.

Electrospun tetracycline (Tet)-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanofibers are considered to have great potential as local drug-delivery systems. This study was designed to explore the effects of the lactidyl/glycolidyl (LA/GA) unit ratio and molecular weight of PLGA on Tet entrapment efficiency and in vitro release kinetics. Three kinds of PLGA (PLGA75/25, M w = 100 000 or 50 000; PLGA50/50, M w = 50 000) were examined in this study. Electrospun nanofibers were fabricated containing 3, 5, 10 wt% Tet. The results showed that PLGA50/50 entrapped more Tet than both PLGA75/25 co-polymers, and the PLGA75/25 of M w = 100 000 entrapped the least amount of Tet, suggesting that the lower the molecular weight of PLGA was, the higher the GA content in PLGA was and the higher the resulting Tet entrapment. Tet loading played an important role in Tet release. Nanofibers with 3 and 5 wt% Tet loading exhibited a sustained release for more than 28 days, whereas 10 wt% Tet only lasted 14 days. Loading of 3 wt% Tet resulted in approx. 35% release in the initial 12 h, 5 wt% Tet released approx. 70% and 10 wt% Tet resulted in approx. 85% release. The integrity of Tet incorporated into electrospun PLGA nanofibers was identified by FT-IR spectrum examination and the bacterial inhibition test. The modified Kirby-Bauer test showed dose-dependent inhibition of Staphylococcus aureus growth by Tet, confirming Tet structural stability throughout the electrospinning procedure. MG-63 cells demonstrated good adhesion and proliferation on all PLGA/Tet fibrous membranes. These results indicate that Tet entrapment and release kinetics of PLGA/Tet composite fibrous scaffolds can be tailored by the LA/GA ratios, molecular weights and drug loadings. Tet-loaded fibrous scaffolds show great potential for local drug delivery and bone defect repair.

In vitro hydrolytic and enzymatic degradation of nestlike-patterned electrospun poly(D,L-lactide-co-glycolide) scaffolds.

A common problem in applying electrospun biodegradable polyester matrixes as tissue-engineering scaffolds is their serious shrinkage with degradation to reduce the porosity drastically. To ameliorate this problem, a nestlike-patterned poly(D,L-lactide-co-glycolide) (PLGA) nanofibrous (∼900 nm) matrix was proposed and fabricated by electropinning. Shrinkage studies demonstrated that the dimension change of nestlike-patterned fibrous membrane was much smaller than those of nonwoven and parallel-aligned fibrous membranes. And the robust framework of the patterned matrix helped to maintain its original nestlike topographical structure during degradation. Compared to hydrolytic-degraded specimens, the PLGA nanofibrous matrixes degraded in the presence of lysozyme showed larger weight loss but slower decrease in molecular weight. Besides, porous fibers with intact surface were detected by scanning electron microscopy after 20-week hydrolysis, and fibers with pores both inside and on surface were observed after enzymatic degradation for 12 weeks. Accordingly, the former presented a bimodal gel permeation chromatography (GPC) peak, while no bi or multimodal GPC peaks were found for the latter as degradation proceeded. These results indicated that an acid autocatalytic effect still existed in the hydrolysis of PLGA nanofibrous matrix. The presence of lysozyme could only accelerate the dissolution of degradation products with low molecular weight, but have no contribution to the chain scission.