OBJECTIVE: To investigate the effects of resveratrol (Res) on aging of marrow mesenchymal stem cells (MSCs), and to explore its mechanism. METHODS: MSCs were isolated from young SD rats and cultured in vitro. The optimal D-gal concentration for induction of MSCs senescence was determined. Then MSCs were randomly divided into four groups, namely the control group, 10μmol/L, 50μmol/L and 100μmol/L Res groups. After the cells were treated with different concentration of Res for 48 h, the senescence-associated changes were examined with senescence-associated-β-galactosidase (SA-β-gal) staining; the expression of p53, p16 and γ-H2AX was evaluated by Western blot. The total active oxygen species (ROS) level was determined by flow cytometry with DCFH-DA staining. In order to assess the effect of Res on the mitochondrial function, MitoSox Red staining was used to detect mitochondrial ROS levels in each group, mitochondrial membrane potential was detected by JC-1 assay, mPTP method was used to detect mitochondrial membrane channel opening level, and Western blot was used to detect the expression level of cytoplasmic cytochrome C (Cyt-C). RESULTS D-gal 10 and 50 g/L significantly increased the number of SA-β-gal positive cells and the level of mitochondrial ROS (all P<0.01). Therefore, 10 g/L D-gal was used to induce the senescence of MSCs in subsequent experiment. Compared with the control group, the number of SA-β-gal positive cells in Res groups significantly decreased (all P<0.01), the expression of p53, p16 and γ-H2AX decreased, and the total and mitochondrial ROS level also decreased (all P<0.01). Moreover, mitochondrial membrane potential, open level of mitochondrial membrane channels and the levels of cytoplasm Cyt-C in the Res treatment groups decreased compared with the control group (P<0.05 or P<0.01). CONCLUSIONS Resveratrol can protect the mitochondrial function of MSCs, and effectively delay the MSC senescence.

Objective:To investigate the dose-effect relationship of Xianfu ointment and its decomposed recipes the 1-chloro-2,4-dini-trochlorobenzene(DNCB) induced chronic eczema in mice, and confirm the median effective dose (ED50) of each formula and the synergetic effect by compatibility. Methods:DNCB was used to induce chronic eczema in C57 mice. The mice were treated with gradient dosages of the Xianfu ointment (11.71-11 662.50 mg?kg-1?d-1,k = 0.316), Anemone flaccid (0.53-530.12 mg?kg-1?d-1,k = 0.316), Xianfu ointment without Anemone flaccid (11.18-11 132.40 mg?kg-1?d-1,k =0.316),respectively. The pathological features were observed after hematoxylin-eosin staining. The volume ratio of epidermides and the number of lymphocyte infiltrated in dermis were analyzed with morphometry. The serum levels of IL-2,IFN-γ,IL-4,and IL-13 were detected by ELISA assay. The ED50was calculated by non-linear regression with various slope using Prism-5.0 software.Results:The effects of Xianfu ointment and its decomposed recipes on chronic eczema showed a dose-dependent tendency. The dose-response curves showed"S"shape. The efficacy of Xianfu ointment on chronic eczema was the most significant among the three formulas, which was demonstrated by decreased epidemical thicknes (ED50= 377.90 mg?kg-1?d-1), reduced infiltrated lymphocyte number(ED50= 153.20 mg?kg-1?d-1), increased serum IL-2(ED50=608.90 mg?kg-1?d-1) and IFN-γ (ED50= 205.50 mg?kg-1?d-1) levels, and decreased serum IL-4(ED50= 198.70 mg?kg-1?d-1) and IL-13 levels (ED50= 117.60 mg?kg-1?d-1). And the dose-effect curves of Anemone flaccid and Xianfu ointment without Anemone flaccid groups were both right shift when compared with that of Xianfu ointment. Conclusion:Xianfu ointment and its decomposed recipes can effectively treat chronic eczema. Anemone flaccid has obvious compatibility synergy in the whole formula. The effects of Xianfu ointment is most significant.

Objective: To study the dose-effect relationship of Yuning ointment and its decomposed recipes in the treatment of oleic acid induced acne in mice. Methods: Oleic acid was administrated to the back (2 cm ×2 cm) of the mice (once a day) for 21 days to induce acne. At d22, the gradient dosage of Anemone flaccida crude drug (1. 06-1 060. 23 mg?kg-1?d-1,k=3. 16), Yuning oint-ment without Anemone flaccida crude drug (4. 73-1 767. 75 mg?kg-1?d-1, k=3. 16) and Yuning ointment (2. 84-2 827. 28 mg?kg-1?d-1, k=3. 16) was respectively administrated to the back of mice for 14 days. The pathological changes of skin were observed by hematoxylin-eosin (HE) staining. The diameter of sebaceous glands and the ratio of follicular keratinization area were morphomet-rically analyzed. The serum levels of IL-1, IL-6 and TNF-α were detected by ELISA assay. The median effective dosages (ED50) of A-nemone flaccida in the three prescriptions were regressed by Prism 5. 01 software to determine the prescription dose-effect. Results: All the therapy groups were with significantly relieved pathological changes of sebaceous glands hypertrophy and follicular keratinization, and decreased serum levels of IL-1, IL-6 and TNF-α in a dose-dependent manner. The dose-response curves showed an "S" shape. A-mong the three therapy groups, the effect of Yuning ointment was the best. The ED50of Yuning ointment regressed by Anemone flaccida dose was 0. 28-fold for improving sebaceous glands hypertrophy, 0. 14-fold for inhibiting follicular keratinization, and 0. 15-, 0. 49-and 0. 24-fold for decreasing serum levels of IL-1, IL-6 and TNF-α. . Regressed by Yuning ointment without Anemone flaccida, the ED50of Yuning ointment was lower than Yuning ointment without Anemone flaccid in terms of improving pathological changes and inhibiting the secretion of cytokines. Conclusion: Yuning ointment can prevent and treat acne through regulating immune function. And the prescrip-tion compatibility can enhance the effects of Anemone flaccida.

Objective: To study the relaxant effect and underlying mechanisms of evodiamine on isolated myometrium of rats. Methods:Prostaglandin F2α( PGF2α) was used to induce isolated myometrium contraction. The relaxant effect of evodiamine and the influence of capsazepine (an antagonist of transient receptor potential cation channel, subfamily V, member 1, TRPV1), U73122 (an antagonist of phospholipase Cβ,PLCβ) and W-7 ( an antagonist of camodulin, CaM) on the relaxant effect of evodiamine on myometri-um were observed respectively by biological function experiments. The median effective concentration ( EC50 ) was analyzed by non-line-ar various slope regressions using Prism-5. 01 software. Results:Evodiamine showed concentration-dependent relaxant effect on PGF2α-induced myometrium contraction with the EC50of 9.56 ×10 -9mol·L-1. Incubation with capsazepine (6.30 ×10 -11 mol·L-1), U73122 (2. 57 × 10 -11 mol·L-1 ) and W-7 (5. 65 × 10 -13 mol·L-1 ) markedly increased the relaxant effect of evodiamine, the EC50 of evodiamine decreased and dose-effect curves left shifted. The order of EC50 was as follows: W-7- evodiamine (8. 88 × 10 -15 mol· L-1) < capsazepine-evodiamine (7.35 ×10 -13 mol·L-1) < U73122-evodiamine (1.95 ×10 -12mol·L-1). Conclusion: Evodia-mine can inhibit myometrium contraction induced by PGF2αobviously, and the mechanisms are probably related to TRPV1, PLCβand CaM.

Objective: To study the relaxant effect and underlying mechanisms of evodiamine on isolated myometrium of rats. Methods:Prostaglandin F2α( PGF2α) was used to induce isolated myometrium contraction. The relaxant effect of evodiamine and the influence of capsazepine (an antagonist of transient receptor potential cation channel, subfamily V, member 1, TRPV1), U73122 (an antagonist of phospholipase Cβ,PLCβ) and W-7 ( an antagonist of camodulin, CaM) on the relaxant effect of evodiamine on myometri-um were observed respectively by biological function experiments. The median effective concentration ( EC50 ) was analyzed by non-line-ar various slope regressions using Prism-5. 01 software. Results:Evodiamine showed concentration-dependent relaxant effect on PGF2α-induced myometrium contraction with the EC50of 9.56 ×10 -9mol·L-1. Incubation with capsazepine (6.30 ×10 -11 mol·L-1), U73122 (2. 57 × 10 -11 mol·L-1 ) and W-7 (5. 65 × 10 -13 mol·L-1 ) markedly increased the relaxant effect of evodiamine, the EC50 of evodiamine decreased and dose-effect curves left shifted. The order of EC50 was as follows: W-7- evodiamine (8. 88 × 10 -15 mol· L-1) < capsazepine-evodiamine (7.35 ×10 -13 mol·L-1) < U73122-evodiamine (1.95 ×10 -12mol·L-1). Conclusion: Evodia-mine can inhibit myometrium contraction induced by PGF2αobviously, and the mechanisms are probably related to TRPV1, PLCβand CaM.

Objective To discuss the influence of Qihong Fang (Wolfberry and Hawthorn Decoction) on heart in aged rats with cold-congealing and blood-stasis pattern.Methods SD rats (n =48) were divided randomly into normal control group,model group,vitamin E group (13.5 mg/kg),low-dose Qihong Fang group (45 mg/kg),mid-dose Qihong Fang group (90 mg/kg) and high-dose Qihong Fang group (180 mg/kg).The aged rat model of cold-congealing and blood-stasis pattern was established through 28-d ozone leading to aging and 14-d ice water leading to cold-congealing and blood-stasis pattern in other 5 groups except of control group.On the 29th d,the blood samples were collected from abdominal aorta for isolating serum and plasma,and heart was separated too.The vitalities of serum total superoxide dismutase (T-SOD),Cu-Zn SOD,Mn-SOD,catalase (CAT),glutathione peroxidease (GSH-PX),content of malondialdehyde (MDA),and prothrombin time (PT),activated partial thromboplastin time (APTT),thrombin time (TT) and fibrinogen (FIB) were detected.The morphologic changes of heart cells were observed after HE staining,fiber deposition quantity was observed after Masson staining,expression of transforming growth factor β1 (TGF-β1) was detected by using immunohistochemistry technique for reviewing aging model of cold-congealing and blood-stasis pattern,and protective effect of Qihong Fang on heart was observed in rats.Results Compared with normal control group,the vitalities of T-SOD,Cu-Zn-SOD,Mn-SOD,CAT and GSH-PX decreased significantly (P ＜ 0.05),MDA content increased significantly (P＜ 0.05,PT and APTT decreased significantly (P ＜ 0.05),and FIB content increased significantly (P ＜0.05) in model group.After Masson staining,morphometry observation by light microscope showed deposition of heart collagen fibers increased significantly (P ＜ 0.05),and expression of TGF-β1 increased significantly (P ＜ 0.05).Compared with control group,the vitalities of T-SOD,Cu-Zn-SOD,CAT and GSH-PX increased significantly (P ＜ 0.05),MDA content decreased significantly (P ＜ 0.05),PT and ATPP increased significantly (P ＜ 0.05),FIB content decreased significantly (P ＜ 0.05),deposition of heart collagen fibers decreased significantly (P ＜0.05),and expression of TGF-β1 decreased significantly (P ＜ 0.05) in mid-dose and high-dose Qihong Fang groups.Conclusion Qihong Fang has a protective effect on heart in aged rats with cold-congealing and blood-stasis pattern.

OBJECTIVETo investigate the effects of D-galactose (D-gal) on aging of rat marrow mesenchymal stem cells (MSCs) and its mechanism.

METHODSMSCs isolated from young (7 d) SD rats were randomly divided into four groups:control group, 1g/L, 10g/L and 50g/L D-gal treatment groups. In control group MSCs were cultured in DMEM containing 10% FBS for 48 h. In the D-gal treatment groups, MSCs were cultured in DMEM containing 10% FBS with 1g/L, 10g/L or 50g/L D-gal for 48 h. The senescence-associated changes were examined with SA-β-galactosidase (SA-β-gal) staining, the expressions of p53, p21 and p16 were detected by Western blot. The living and apoptotic cells were determined by AO/EB staining. Cell proliferation was detected by MTT assay. SOD activity was measured by xanthine oxidase method, and the MDA content was estimated with thiobarbituric acid (TBA) method.

RESULTSCompared to control group, the number of SA-β-gal positive cells and the expression of p53, p21 and p16 were significantly increased in the 10g/L and 50g/L D-gal treatment groups. The apoptosis rate in 50g/L D-gal group was significantly higher than that in control group (P<0.01). The proliferation of MSCs was decreased in the 10g/L and 50g/L D-gal groups compared to control group (P<0.05). After 10g/L and 50g/L D-gal treatment, SOD activity was significantly decreased (P<0.01), and MDA level was increased (P<0.01).

CONCLUSIONThe aging of MSCs can be induced by 10g/L and 50g/L D-gal, which may be associated with the elevated levels of oxidative stress.

Animals ; Apoptosis ; drug effects ; Cells, Cultured ; Cellular Senescence ; drug effects ; Galactose ; pharmacology ; Male ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley

For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages, RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western blotting. The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line. These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.
Animals ; Antigens, Bacterial ; biosynthesis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; Cell Line ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Macrophages ; cytology ; metabolism ; Mice ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Objective To investigate the effect of Xuebi-jing injection on the change of the serum high-sensitivity C-reactive protein (hs-CRP) in patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD). Methods 86 patients with AECOPD were enrolled in this study, who was randomly divided into a control group (43 cases)and a treatment group (43 cases). Both treatment group and control group were given conventional therapy. On this basis, Xuebi-jing injection was added to the treatment group. Hs-CRP, blood gas analysis, blood cell count and severity of illness score (acute physiological and chronic health evaluation Ⅱ, APACHE Ⅱ) were monitored before and 5 days after treatment. Results There was no obvious difference of hs-CRP levels between two groups before treatment. Hs-CRP was decreased in both groups after the treatment, while the treatment group showed an obvious decrease compared with the control group (t＝5.3302,P＜0.01). Blood gas analysis, blood cecount, APACHE Ⅱ scores were improved after the treatment in both groups, while the improvement in treatment group showed obvious difference than the control group (t＝13.5089、26.3077 、13.4053,P＜0.01). Conclusion The conventional therapy plus Xuebi-jing injectiong for AECOPD can improve serum hs-CRP levels and improve the efficacy of AECOPD .

Objective To observe the effect of digestive load from senna on the adaptive thermogenesis in hypothyroid rats.Methods After being born(d1),rats were given the intraperitoneal injection with a series ratio of 131Ⅰ at neutral temperature 28℃ and a graded developmental hypothyrosis was resulted in.Since d56,they were fed with higher lipid 24 h then fasted 24 h interval to make them sensitive to digestive load.Since d70,senno sides were given intragastrical administration for 14 d to resulted in the digestive load with its empting function of the bowels.Pre-(d67-69)and post-(d85-86)administration of senna under the neutral environmental temperature,all changes of rat temperature,included peaks(℃)and areas under curve(℃?h),were measured and recorded with physiography,during 180 min after being injected with 106 ?g?kg-1 isoprenaline intravenously.The correlation between the effect and the logarithmic dose of 131Ⅰ(nCi?g-1)was analyzed.As well as EC50,the decreased thermogenesis induced by 131Ⅰ,was calculated with peaks and areas under curve respectively.The correlative analysis between the digestive load and the adaptive thermogenesis was performed especially with senna.Results Before and after administrated senna,it was negative correlation between rat temperature change(peaks and areas under curve)induced with isoprenaline and the degree of hypothyrodism(P