D-Optimal Design and Development of a Koumine-Loaded Microemulsion for Rheumatoid Arthritis Treatment: In vivo and in vitro Evaluation.

Introduction: Koumine (KME) is the most abundant active ingredient separated from Gelsemium elegans Benth and exhibits a significant therapeutic effect on rheumatoid arthritis (RA). It is a lipophilic compound with poor aqueous solubility, and there is an urgent need to develop novel dosage forms of KME and promote its clinical application for the treatment of RA. The aim of this study was to design and develop KME-loaded microemulsions (KME-MEs) for the effective management of RA. Methods: The composition of the microemulsion was selected by carrying out a solubility study and generating pseudoternary phase diagrams, and further optimized by D-Optimal design. The optimized KME-MEs was evaluated for particle size, viscosity, drug release, storage stability, cytotoxicity, cellular uptake, Caco-2 cell transport and everted gut sac investigations. In vivo fluorescence imaging and the therapeutic effects of KME and KME-MEs on collagen-induced arthritis (CIA) rats were also evaluated. Results: The optimized microemulsion contained 8% oil, 32% Smix (surfactant/cosurfactant) and 60% water and was used for in vivo and in vitro studies. The optimal KME-MEs exhibited a small globule size of 18.5 ± 0.14 nm and good stability over 3 months, and the release kinetics followed a first-order model. These KME-MEs had no toxic effect on Caco-2 cells but were efficiently internalized into the cytoplasm. Compared to KME, the KME-MEs displayed significantly increased permeability and absorption in Caco-2 cell monolayer assay and ex vivo everted gut sac experiment. As expected, the KME-MEs attenuated the progression of RA in CIA rats and were more effective than free KME with a reduced frequency of administration. Conclusion: The KME-MEs improved the solubility and therapeutic efficacy of KME by employing formulation technology. These results provide a promising vehicle for the oral delivery of KME to treat RA and have attractive potential for clinical translation.

[Genetic Polymorphism of Antigens in Twelve Rare Blood Group Systems of Li Nationality in Hainan Province].

OBJECTIVE: To explore the distribution characteristics of main antigen gene frequencies of Duffy,Diego,Kidd,Dombrock,MNS,Lutheran,Kell,Colton,Scianna,Yt,Knops and Indian in red blood cell blood group system of Li nationality in Hainan Province. METHODS: Antigens in twelve rare blood group systems of 214 Li people in Hainan Province were genotyped and analyzed by polymerase chain reaction-sequence specific primers (PCR-SSP). RESULTS: The gene frequency of antigens in twelve rare blood group systems of 214 Li people in Hainan Province including: the gene frequency of Duffy blood group system: fya=0.9556，fyb=0.0444；the gene frequency of Diego blood group system: Dia=0.0678，Dib=0.9322；the gene frequency of Kidd blood group system：JKa=0.4533，JKb=0.5467；the gene frequency of Dombrock blood group system：DOa=0.1051，DOb=0.8949；the gene frequency of MNS blood group system：M=0.8131，N=0.1869,S=0.0327，s=0.9673，Mur+=0.5748，Mur-=0.4252；the gene frequency of Lutheran blood group system：AUa=0.8318，AUb=0.1682；the Kell, Colton, Scianna, Yt, Knops and Indian blood type systems showed a monomorph and the genotype was kk, coacoa, Sc1Sc1, YtaYta, KnaKna and InbInb. The observed and expected genotype values in twelve rare blood group systems of Li nationality in Hainan province were obeyed by the Hardy-Weinberg genitic rules. The difference between the observed and expectated values of the Kidd blood group system showed significant differences（χ2=17.1946，P=0.0002）. CONCLUSION: The genetic distribution and genetic status in twelve rare blood group systems of Li nationality in Hainan Province are relatively stable. The gene distribution of Duffy, Diego, Kidd, Drombrock, MNS and Lutheran blood group systems are polymorphic and show unique distribution characteristics compared with other regions and different nationalities. The gene frequency distribution of Kell、Colton、Scianna、Yt、Knops、Indian blood group systems are monomorphic.

Study on the compatibility effect and active constituents of Atractylodis Rhizoma in Ermiao Wan against Acute Gouty Arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ermiao Wan (EMW), composed of Atractylodis Rhizoma (AR) and Phellodendri Chinensis Cortex (PC), is a classical traditional Chinese medicine prescription having been used to treat the disease named "Tong Feng", which is described as "ache in bones and joints" with the same symptom of modern disease named acute gouty arthritis for many years in TCM clinical practice. Besides, both PC and AR were considered to be effective in anti-inflammatory according to modern pharmacological research. AIM OF THE STUDY: Present study was undertaken to probe the compatibility rationality between the two herbs PC and AR in EMW and the active constituents of AR against acute gouty arthritis (AGA). MATERIALS AND METHODS: Rat model of AGA was induced by intra-articular injection of monosodium urate (MSU) crystal suspension, and PC combined with or without different AR extracts were used for AGA treatment. Ankle joint swelling, proinflammatory cytokines in serum and pathological changes of synovium were investigated. Using the developed UHPLC-QQQ-MS method, the plasma concentrations of the primary alkaloids in PC, such as berberine, phellodendrine, magnoflorine, jatrorrhizine, berberrubine, palmatine, and tetrahydropalmatine, in AGA rat were determined, and pharmacokinetics properties were compared following oral administration of PC, PC combined with or without different AR extracts. RESULTS: PC, PC combined with AR volatile oil (VO) extract or PC combined with whole AR extract significantly attenuated the ankle joint swelling of AGA rats. Besides, the combination of PC and VO extract of AR showed superior efficacy than other groups in ameliorating ankle joint swelling, reducing the IL-6 expression in serum and improving tissue lesions of ankle joints. Furthermore, it turned out that the VO extract of AR increased the blood exposure level of PC related alkaloids than non-volatile oil (NVO) extract of AR, by comparing the pharmacokinetic results of each group. CONCLUSIONS: The VO components of AR were the key compatible materials to combine with PC in EMW for AGA treatment. Moreover, the enhanced anti-AGA activity of PC after combining with VO extract of AR may attribute to the influence of VO on the pharmacokinetics of PC. This study may provide useful information for elucidating the compatibility effects of AR in EMW against AGA.

[Short-term response of carbon emission to snow cover change in Calamagrostis angustifolia wetlands of Sanjiang Plain, Northeast China]. / ä¸‰æ±Ÿå¹³åŽŸå°å¶ç« æ¹¿åœ°ç¢³æŽ’æ”¾å¯¹é›ªè¢«å˜åŒ–çš„çŸ­æœŸå“åº”.

To understand the response of Calamagrostis angustifolia wetland of the Sanjiang Plain to changes in snow cover, we examined the greenhouse gases emission flux of the removed snow treatment (0 cm, RS), the added snow treatment (50 cm, AS) and the control (20 cm, CK) of a C. angustifolia wetland, and their relations with environmental factors with the method of the static chamber-gas chromatography. The results showed that soil temperature, soil water content, and carbon emissions were lowest during the snow-covering period under all treatments, and gradually increased with time. With the increases of time and snow thickness, soil temperature was rised and the difference of three treatments gradually was decreased. Soil water content of RS was always lower than that of CK and AS. AS and CK could promote soil CO2 emission compared with RS during and after snowmelt. The soil cumulative CH4 emissions differed little among the treatments. There was significant correlation between soil temperature and cumulative CO2 and CH4 emissions. With the increases of soil temperature, soil cumulative CO2 emission continued to increase and soil cumulative CH4 emission decreased firstly and then increased rapidly. Soil water content was significantly correlated with cumulative CO2 and CH4 emissions. As the soil moisture increased, the cumulative soil CO2 emission gradually increased, reaching a certain threshold and then flattening, while soil cumulative CH4 emission continuously increased.

[Fungal Community Structure of Different Degeneration Deyeuxia angustifolia Wetlands in Sanjiang Plain].

To understand the wetland soil fungal community structure and diversity in different degeneration Deyeuxia angustifolia wetlands, the topsoil (0-20) of three different degeneration D. angustifolia wetlands were collected in the Sanjiang Plain field experiment station of the Institute of Nature and Ecology, Heilongjiang Academy of Sciences. The distribution and variation of soil fungal diversity were assessed by high-throughput sequencing method. The results showed that Shannon-Wiener index increased from marsh Deyeuxia angustifolia wetland marsh meadow Deyeuxia angustifolia wetland meadow Deyeuxia angustifolia wetland. Sequence blast showed that the fungal taxonomy belonged to Ascomycota, Basidiomycota, Chytridiomycota, Fungi_unclassified, Zygomycota, which dominant fungi were Fungi_unclassified (75.12%),Ascomycetes (56. 56%), Basidiomycetes (72.65%) in the three degeneration wetlands, respectively. The fungal structure compositions and diversities of marsh meadow Deyeuxia angustifolia wetland and meadow Deyeuxia angustifolia wetland were similar according to Heatmap analysis. The fungal community structure was influenced by soil nutrients (explained 88.62%) and plant composition (explained 9.85%) through the Variation partition analysis (VPA). In conclusion, the fungal community structure was significantly different, which was influenced by soil water content, in different degeneration Deyeuxia angustifolia wetlands in Sanjiang plain. The results may supply scientific basis for studying fungal diversity and spatial heterogeneity in degeneration wetlands.

Evodiamine Suppresses ABCG2 Mediated Drug Resistance by Inhibiting p50/p65 NF-κB Pathway in Colorectal Cancer.

Evodiamine (Evo), extracted from the Chinese herbal medicine Evodia rutaecarpa, has cytotoxic effects on different types of human cancer cells. However, its effects on drug resistance and their molecular mechanism and therapeutic target in colorectal cancer are not well understood. In the present study, we observed that Evo inhibited cell growth and induced apoptosis in adose-and time-dependent manner in HCT-116/L-OHP cells. Moreover, Evo treatment reduced Rhodamine 123 accumulation and ATPase activity in HCT-116/L-OHP cells, indicating that Evo decreased the efflux function in HCT-116/L-OHP cells. Interestingly, phosphorylation of NF-κB pathway, particularly p50/p65, was also inhibited by Evo treatment. Furthermore the effect of Evo in reversing drug resistance and suppressing phosphorylation of NF-κB pathway were attenuated after treatment with the NF-κB activator (LPS). Additionally, Evo inhibited the tumor growth in a colorectal MDR cancer xenograft model and down regulated p-NF-κB level in vivo. Our study provided the first direct evidence that Evo can attenuate multidrug resistance by blocking p-NF-κB signaling pathway in human colorectal cancer. Evo could be a potential candidate for cancer chemotherapy.

Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway.

BACKGROUND: Zuo-Jin-Wan (ZJW), a traditional Chinese medicine formula, has been identified to be effective against drug resistance in cancer. In the present study, we investigated the effect of ZJW on acquired oxaliplatin-resistant and the PI3K/Akt/NF-κB pathway in vitro. METHODS: We tested the dose-response relationship of ZJW on reversing drug-resistance by CCK-8 assay and flow cytometry analysis in vitro. The protein expression of P-gp, MRP-2, LRP, and ABCB1 mRNA expression level were evaluated by Western blot and quantitative RT-PCR. The activities of PI3K/Akt/NF-κB pathway were also examined with or without ZJW, including Akt, IκB, p65 and their phosphorylation expression. RESULTS: We found that ZJW significantly enhanced the sensitivity of chemotherapeutic drugs and increased oxaliplatin (L-OHP)-induced cell apoptosis in a time- and dose-dependent manner. Moreover, both ZJW and a PI3K specific inhibitor (LY294002) suppressed phosphorylation of Akt (Ser473) and NF-κB, which is necessary in the activation of the PI3K/Akt/NF-κB pathway. The effect of ZJW in reversing drug-resistance and suppressing phosphorylation of Akt (Ser473) and NF-κB were weakened after treatment with a PI3K/Akt activator in HCT116/L-OHP cells. CONCLUSIONS: Our study has provided the first direct evidence that ZJW reverses drug-resistance in human colorectal cancer by blocking the PI3K/Akt/NF-κB signaling pathway, and could be considered as a useful drug for cancer therapy.

[Roles of CD8+ CD28- T regulatory cells in acute infectious mononucleosis in children].

OBJECTIVE: To explore the role of CD(8)(+)CD(28)(-) T regulatory cells (Tr) in the immunological pathogenesis of acute infection with Epstein-Barr virus in children. METHODS: The present study enrolled 25 children with infectious mononucleosis (IM) and 25 age-matched healthy children. Flow cytometric analysis was performed to detect the percentage of CD(3)(+), CD(3)(+)CD(4)(+), CD(3)(+)CD(8)(+), CD(8)(+)CD(28)(+) by determining the ratio of positive cells in lymphocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR were used to analyze IL-6, IL-10, IFN-gamma expression in CD(8)(+)CD(28)(-) Tr cells and ILT-3, ILT-4 expression in monocytes/macrophages. RESULTS: The proportions of CD(8)(+)CD(28)(-)T cells in children with acute-phase IM was significantly higher than those in the controls (P < 0.01). The expression level of IL-6, IL-10, IFN-gamma, ILT-3, ILT-4 mRNA significantly increased compared to those of the controls (P < 0.01). CONCLUSION: The CD(28) expressed on CD(8)(+) T cells in vivo is gradually lost with age and CD(8)(+)CD(28)(-) cells increase up 50% to adult. EBV can directly infect B cells, trigger CD(8)(+) CTL response and destroy the target cells to cause serious immunopathological lesion. Therefore we speculate that the expansion of CD(8)(+)CD(28)(-) Tr cells in children with IM may be an adaptive immune response to avoid serious inflammation and autoimmune reactions.

[The role of CD4+ CD25+ regulatory T cells in the pathogenesis of asthma in children].

OBJECTIVE: To investigate the role of CD4(+)CD25(+) regulatory T cells (Tr cells) in the pathogenesis of asthma in children. METHODS: Peripheral blood samples were collected from 20 pediatric patients with asthma, 10 male and 10 female, aged 7 (3-12), and 20 healthy children, 10 male and 10 female, aged 6.5 (2-11). Lymphocytes were isolated. Flow cytometry was used to examine the percentages of CD4(+)CD25(+) regulatory T cells, IL-10 secreting CD4(+)CD25(+) regulatory T cells (CD4(+)CD25(+)-IL-10), and transforming growth factor (TGF)-beta secreting CD4(+)CD25(+) regulatory T cells (CD4(+)CD25(+)-TGF-beta). RT-PCR and real-time PCR were used to detect the mRNA expression of suppressor of cytokine signal 1 (SOSC1) and Foxp3. RESULTS: The percentages of CD4(+)CD25(+) regulatory T cells of the asthma children was 6.51% +/- 1.94%, significantly lower than that of the healthy children (11.96% +/- 2.30%, P < 0.01); the percentage of CD4(+)CD25(+)-IL-10 of the asthma children was 1.46% +/- 0.35%, significantly lower than that of the healthy children (5.65% +/- 1.70%, P < 0.01); and the percentage of CD4(+)CD25(+)-TGF-beta of the asthma children was 1.24% +/- 0.21%, significantly lower than that t of the healthy children (4.23% +/- 1.65%, P < 0.01). The Foxp3 mRNA expression of the asthma children was 0.12 +/- 0.05, significantly lower than that of the healthy children (1.71 +/- 0.58, P < 0.01); and the SOCS1 mRNA expression of the asthma children was 0.38 +/- 0.19, significantly lower than that of the healthy children (1.51 +/- 0.41, P < 0.01). CONCLUSION: The decrease of CD4(+)CD25(+) regulatory T cells may be involved in the pathogenesis of asthma. The decreased mRNA expression of Foxp3 and SOCS1 may be associated with the aberrant development of CD4(+)CD25(+) regulatory T cells.