Aim To investigate endoplasmic reticulum stress ( ERS)-mediated high-fat diet and palmitic acid-induced insulin resistance ( IR) in skeletal muscle and interventional effects of fenofibrate both in vivo and in vitro tests. Methods Female SD rats were randomly subjected to a standard control diet ( SCD) or high-fat diet ( HFD) for 20 weeks, then the HFD group was di-vided into high-fat-diet group and high-fat-diet group treated with fenofibrate ( HFD +FF, 30 mg · kg-1 · d-1 ) for another 8 weeks. The changes of IR and ex-pression of peroxisome proliferator-activated receptor α( PPARα) , glucose regulated protein 78 ( GRP78 ) and transcription factors GADD153 ( CHOP ) were as-sessed respectively. C2C12 myotubes were divided into normal control group ( NC ) , model group ( palmitic acid, PA) , postive control drug group ( tunicamycin, TM) and treatment group ( fenofibric acid, FA+PA) , the expressions of GRP78 and CHOP were assessed re-spectively. Insulin-stimulated phosphorylation of Akt was also analyzed to detect changes of insulin sensitivi-ty in C2 C12 . Results The high-fat diet induced obvi-ous IR and upregulated ERS markers GRP78 and CHOP in skeletal muscle of rats, and these responses were attenuated by treatment with fenofibrate. Incuba-tion of myotubes with palmitic acid or tunicamycin sig-nificantly increased expression of ERS markers GRP78 and CHOP. Meanwhile, insulin-stimulated phosphoryl-ation of Akt was inhibited obviously. Pre-incubation with FA markedly inverted PA-induced ERS and insu-lin-stimulated phosphorylation of Akt. Conclusion Fenofibrate ( fenofibric acid) has obvious effects of IR on skeletal muscle tissues and cells, which may be re-lated with reduced expression of GRP78 and CHOP in ERS.

BACKGROUND:Systemic lupus erythematosus (SLE) is classified into four types, and the major treatment is to tonify kidney and nourishyin, clear blood stasis and toxin by the traditional Chinese medicine (TCM). Even though, there are stil many patients with poor efficacy. Mesenchymal stem cels have the capacity of multiple differentiation, hematopoietic support and immune regulation, thus having been used for the treatment of refractory, recurrent SLE and achieving good effects. OBJECTIVE:To investigate the therapeutic effect of umbilical cord-derived mesenchymal stem cel transplantation on SLE patients with different patterns of syndromes. METHODS: Twenty-one SLE patients were clustered to four syndrome types of TCM, including heat-toxin,yin deficiency of liver and kidney,yang deficiency of spleen and kidney, andqi stagnation and blood stasis. The changes in clinical and laboratory indicators were analyzed statisticaly before and after cel transplantation. RESULTS AND CONCLUSION:The level of 24-hour proteinuria and SLE disease activity index scores in SLE patients were significantly decreased at 1, 3, 6 months after cel transplantation (P < 0.01). Umbilical cord-derived mesenchymal stem cel transplantation could significantly reduce the 24-hour proteinuria in SLE patients withyin deficiency of liver and kidney at 1, 3 and 6 months (P < 0.01), while slightly reduce the 24-hour proteinuria in SLE patients with heat-toxin andqi stagnation and blood stasis at 1, 3 months (P < 0.05) as wel as in SLE patients withyang deficiency of spleen and kidney at 1 month (P < 0.05). Additionaly, umbilical cord-derived mesenchymal stem cel transplantation could increase the serum albumin levels in al the SLE patients (P < 0.01), although the changes in patients with heat-toxin were moderate (P < 0.05). Al the SLE patients of four types had an increasing trend of their platelet counting after cel transplantation, but there was no statistical difference before and after cel transplantation. Taken together, umbilical cord-derived mesenchymal stem cel transplantation is effective for treatment of SLE, but has different therapeutic efficacy on SLE patients with different syndrome types of TCM.

Objective To investigate whether activation of protein kmase C (PKC) can induce the activation and nuclear translocation of nuclear factor enthroid 2-related factor 2 (Nrf2) in retinal pigment epithelial (RPE) cells in vitro,and explore whether PKC activation may affect the expression of Nrf2 in RPE cells.Methods PKC-specific activator phorbol ester PMA was used to culture rabbit RPE cells and RPE cells pretreated with Nrf2 inhibitor for 24 hours.Immunofluorescence and Western blot were used to detect Nrf2 in the nucleus of the expression of the situation,the data were obtained for statistical analysis.Results The expression of Nrf2 protein in the nucleus of PRE cells was detected by immunofluorescence.Compared with the control group,the expression of Nrf2 protein in the nucleus of RPE cells increased in the experimental group,and the increase of PMA + Nrf2 inhibitor group was lower than that of PMA group.The difference between the two groups was statistically significant (P ＜0.05).Western blot analysis showed that the Nrf2 protein in the nucleus of PRE was quantitatively analyzed by image analysis.The gray value of the control group was significantly different (0.286 ± 0.013 in the control group,1.304 ± 0.033 in the PMA group and 0.671 ± 0.087 in the PMA + Nrf2 inhibitor group,P ＜ 0.05).Conclusion PKC can activate nuclear translocation of Nrf2 in rabbit RPE cytoplasm,and Nrf2 inhibitor can attenuate the effect of PKC.

Objective To investigate the immunoregulatory effect of thalidomide on the peripheral blood T-iymphocytes in systemic lupus erythematosus patients in vitro. Methods T-lymphoeytes were treated by thalidomide with different concentrations, then the proliferation of these T-lymphocytes proliferation was deteted by MTT while apoptosis and lymphocyte activation marker were analyzed by flow cytometry. The mRNA expression of IL-6, IL-10 and TNF-α was measured by real-time RT-PCR, One-way ANOVA was used for statistical analysis. Results In vitro, thalidomide inhibited the expression of CD3~+CD28~+ [500 μg/ml group vs the control group, (48±9)% vs (57±9)% P<0.05]. The pro-portion of apoptotic T-lymphoeytes in the 500 μg/ml group was (36±8)%, which was significantly higher than that in the control group [(23±5)%,P<0.05 ]. The values of A_(570nm) T-lymphocytes were significantly lower in the 100 μg/ml group, 300 μg/ml groupand 500 μg/ml group compared with those of the control group ( 100 μg/ml group vs 300 μg/ml group vs 500μg/ml group vs the control group, 0.39±0.05 vs 0.34±0.04 vs 0.30±0.03 vs 0.51±0.07, P<0.05), while thalidomide promoted the expression of CD8~+CD152~+ [ 100 μg/ml group vs 500 μg/ml group vs the control group, (5.0±0.6)% vs (7.8±0.7)% vs (4.2±0.6)%, P<0.05 ]. 500 μg/ml thalidomide inhibited the mRNA expression of IL-6, 2.5～500 μg/ml thalidomide inhibited IL-10, TNF-α mRNA expression of T-lymphocytes.Conclusion Thalidomide can inhibit the proliferative activities and CD28 expression of T-lymphocytes,reduce mRNA expression of IL-6, IL-10, TNF-α, stimulate CD28 expression and apoptosis of T-lymphocytes. These effects may play an important role in it's immune-suppressive effects on systemic lupus erythematosus.

Objective To study the expression and significance of Th17 cells from peripheral blood of patients with rheumatoid arthritis (RA). Methods Intracelluar flow cytomete detection of IL-17/IFN-γ and IL-17/IL-6 was established using anti-CD3/Anti-CD28/IL-23 as stimulators after isolation of untouched human CD4~+T cells from PBMC. There were three groups in the present study: ①healthy controls group; ② RA stable group; ③RA active group. Results The isolation of untouched human CD4~+T cells from PBMC was effective and its purity was over 90%. The percentage of intracelluar IL-17 in CD4~+ T cells from RA patients was increased significantly. Such percentage in active group (1.54±0.41) was higher than that of stable group (0.70±0.21, P<0.01) and both of them were higher than those of healthy controls (0.42±0.12, P<0.01). Under anti-CD3/Anti-CD28/IL-23 stimulation, the percentage of intracelluar IL-17 was also increased significantly(P<0.01). The porcentage of intracellular IFN-γ was similar to that of IL-17, while that of IL-6 was not significantly different. There is an correlation between IL-17 and IFN-γ or IL-6. Conclusion There is an abnormal expression of IL-17 and IFN-γ in human CD4~+T cells in RA patients, which is related to disease activity . Th17 cells may be used as a new marker for the assessment of RA activity.

Objective To investigate the immunoregulatory effects of mesenchymal stem cells (MSCs)on peripheral blood T lymphocytes from patients with systemic lupus erythematosus(SLE)in vitro and their potential mechanism.Methods MSCs were isolated from the bone marrow of 3 healthy human volunteers,cultivated and identified.Under phytohemagglutinin(PHA)stimulating,peripheral blood T lymphocytes from 8 patients with SLE were treated with MSCs with the T lymphocyte/MSC ratio being 50:1 in group B and 5:1 in group C or without MSCs(group A).MTT assay was used to detect the proliferation of T lymphocytes.flow cytometry to analyze the expressions of surface markers CD152 and CD28 on T lymphocytes.and real time PCR to measure the mRNA expressions of interleukin-6 and interferon-γ,in the T lymphocytes.Results MSCs could markedly inhibit the proliferation of T lymphocytcs.The proliferation of T lymphocytes expressed as absorbance value at 570 nm was 0.484±0.032 in group B.0.308±0.025 in group C,significantly lower than that in group A(0.765±0.036,both P＜0.05),and significant difference was also observed between group C and B(P＜0.05).In the case of the percentage of CD28 positive T lymphocytes.group B and C were significantly lower than group A(60.39%±3.92%and 45.05%±3.46%vs 74.73%±3.74%,both P＜0.05),and group B significantly differed from group C(P＜0.05).MSCs had no obvious effect on the expression of CD152 on T lymphocytes,but significantly suppressed the mRNA expression of interleukin-6 and interferon-γ(both P＜0.05).and the suppressive effect was enhanced with the incrgase in MSC count.ConclusionsMSCs exert an immunosuppressive effect on T lymphocytes from patients with SLE,likely through inhibiting the proliferation,CD28 expression,interleukin-6 and interferon-γ mRNA expression of T lymphocytes.

Objective To investigate the mechanism of tumor necrosis factor-α (TNF)-α inhibiting osteo blastdifferentiation of mesenchymal stem cells (BMMSCs) in the pathogenesis of osteoporosis in the mouse model of systemic lupus erythematosus (MRL/lpr). Methods The femurs of MRL / lpr and C3He/HeJ mice were isolated, the bone structure were examined by hematoxylin-eosin (HE) staining. The proteins of TNF-α, NF-κB P50, bone morphogenetic protein -2 (BMP-2) and PSmad1/5/8 were measured by immunohistochemical stain. Bone marrow mesenchymal stem cells (BMMSCs) were isolated. After BMMSCs grew on the cover slips, the proteins on top of it were evaluated by immunohistochemistry stain. Moreover, the alkaline phosphatase (ALP) staining was employed for the measurement of the early osteogenic differentiation. BMMSCs together with hydroxyapatite were embedded subcutaneously in the nude mice and eight weeks later, the ectopic bone formation was evaluated. The recombinant human tumor necrosis factor receptor type Ⅱantibody fusion protein (etanercept) or normal saline was subcutaneous injected to the mice with lupus. After four weeks, the expression of these proteins was observed and the ectopic bone formation was investigated. Image-Pro plus 6.0 software was employed for imagine analysis, and Studentˊs t-test was used to test the differences between 2 independent groups. Results MRL/lpr mice showed decreased volume of cortex and the percentage of cortex to the volume of bone of MRL/lpr mice was significantly lower compared to control groups and with C3He/HeJ mice (13.96±0.25 vs 23.61±0.71, n=3, P<0.01). The protein levels of both TNF-αand NF-κB P50 on the femur of MRL/lprl mice were higher than those of the control group (0.643±0.051 vs 0.405±0.022, 0.917±0.023 vs 0.650±0.032, n=3, P<0.01). The expressions of BMP-2 on the femur of MRL/lpr mice were lower than those of the C3He/HeJ mice (0.52 ±0.03 vs 0.72 ±0.03, n=3, P<0.01). There was no difference in the expression of PSmad1/5/8 on the femur between the two groups by immunohistochemistry detection (1.264 ±0.021 vs 1.301± 0.044, n=3, P>0.05). The expressions of TNF-α and NF-κB P50 in BMMSCs of MRL/lprl mice were higher than those of the C3He/HeJ (0.184±0.021 vs 0.136±0.013, 0.132±0.021 vs 0.097± 0.014, n=3, P<0.01), while BMP-2 and PSmad were lower than those of the control group (0.128±0.013 vs 0.216±0.221, 0.115±0.023 vs 0.196±0.034, n=3, P<0.01). After 7 days of BMP-2 stimulation, the activities of ALP of BMMSCs from MRL/lprl mice were reduced detected by ALP staining and the osteoblast differentiation of these cells were decreased than BMMSCs from the control mice by HE and Masson staining. The percentage of the cortex to the volume of bone of the etanercept injection MRL/lpr mice was higher than that of the control group (21.8±1.0 vs 14.3 ±0.6, n=3, P<0.01). Moreover, the proteins of TNF-α and NF-κB P50 on the femurs of such injected mice were lower than those of the control group (0.540±0.024 vs 0.682±0.031, 0.857±0.023 vs 1.098±0.044, n=3, P<0.05), while the expressions of BMP-2 were higher than the control group (0.99±0.04 vs 0.85±0.04, n=3, P<0.05). There was no difference in the PSmad1/5/8 expression on the bone of the two group of lupus mice (0.88 ±0.08 vs 0.84 ±0.04, n=3, P>0.05). The ectopic bone formation of BMMSCs of the etanercept injected MRL/lpr mice was higher than that of the normal saline injected mice, however, it was lower than that of the C3He/HeJ mice. Conclusion TNF-α inhibits osteoblast differentiation of mesenchymal stem cells by depressing Smad signaling which may contribute to the osteoporosis of the lupus mice.

PurposeThe bone flare phenomena has been well described on bone scintigraphy for efficacy monitoring up to now, but our knowledge has been rarely described on MSCT, the phenomena may be erroneously classiifed as disease progression. This article intends to evaluate the existence and CT features of bone flare phenomena of metastatic bone disease in lung cancer patients treated with ibandronate, to raise awareness of this phenomenon.Materials and Methods The clinical and image data of 45 patients with bone metastases of lung cancer were retrospectively analyzed prior to treatment and 3, 6 months after treatment, the change of CT value and CT features 3 months after treatment between bone flare phenomena group, progressive disease group 1 and progressive disease group 2 were compared.Results The incidence of bone flare phenomena was 6.7% (3/45). 3 months after treatment, CT value of the bone flare phenomena group and progressive disease group 1 changed when compared with before treatment, the differences were statistically signiifcant (t=-5.787 and-2.788,P<0.05) and there was no statistically signiifcant difference (t=1.691,P>0.05) of CT value in the progressive disease group 2 after 3 months' treatment. After 3 months' treatment, the bone flare phenomena group mostly appeared as osteogenic sclerosis of osteolytic lesions, while the cases of progressive disease group mostly appeared as new periosteal reaction of the lesion, or osteogenic/mixed lesion combined with osteolytic damages, the difference between the two groups was statistically signiifcant (χ2=10.139, 8.041 and 4.154,P<0.01,P<0.05). There was no statistically signiifcant difference in increase of density in osteosclerotic lesions (χ2=0.059, P>0.05).Conclusion In patients treated with ibandronate, when there is therapeutic effect evaluation standard of bone metastases (disease progression) and clinical comprehensive curative effect evaluation standard (effective) discordance at 3 months after treatment, it can be interpreted as bone flare phenomena, and the change of CT features contributes to the differential diagnosis of bone flare phenomena with progressive disease.

Objective To investigate the protective effect of mild hypothermia on cerebral ischemia-reperfusion injury in rats and the effect of mild hypothermia on the expression of inhibitor of differentiation 2 (Id2) protein.Methods A total of 72 adult male rats were randomly divided into a sham operation group,a normothermia group,and a mild hypothermia group.A model of middle cerebral artery occlusion was induced by a suture method.The mild hypothermia group was treated with low temperature (anal temperature 33±1 ℃,tympanic membrane temperature 31±1 ℃).Modified Neurological Severity Score (mNSS) was used to evaluate neurological deficits,triphenyltetrazolium chloride staining was used to detect infarct volume,and Western blot was used to detect the Id2 expression in the ischemic cortex at ischemia-reperfusion 6,12,24,and 72 h,respectively.ResultsThe mNSS scores in the mild hypothermia group were significantly lower than those in the normothermia group,the infarct volumes were significantly smaller than those in the normothermia group at ischemia-reperfusion 6,12,24,and 72 h (all P<0.001).Western blot analysis showed that the Id2 expressions in the ischemic cortex in the mild hypothermia group were significantly lower than those in the normothermia group at ischemia-reperfusion 6,12,24,and 72 h (all P<0.05).Conclusion s Mild hypothermia can decrease neurological deficits and reduce infarct volume after cerebral ischemia-reperfusion,its mechanism may be associated with the down-regulation of the Id2 expression.

Objective To investigate the immunoregulatory effects of mesenchymal stem cells (MSCs) on the peripheral T-lymphocytes of lupus nephritis in vitro. Methods MSCs were isolated and expanded from human bone marrow cells. The purity of MSCs was identified by flow cytometry (FCM). The MSCs (4×104, 1×104, 2×103) were added into wells containing peripheral blood lymphocytes (2×105) from lupus nephritis in the presence of phytohemagglutinin [PHA). Samples were divided into the following groups: group A:T-lymphocytes alone; group B: MSCsl with T-lymphocytes(MSCsl:T=1:5); group C: MSCs2 with T-lymphocytes (MSCs2:T=1:20); group D: MSCs3 with T-lymphocytes (MSCs3:T=1:100). The proliferation of T-lymphocytes was assessed by MTT. FCM was used to analyze the apoptosis of T-lymphocytes and surface markers of CD28 and CD152. The gene expression of interferon γ (IFN-γ), interleukin-10 (IL-10), transforming growth factor-β1 (TGF-β1) were detected by real-time RT-PCR. Results In vitro, with the presence of MSCs, peripheral blood T-lymphocytes from lupus nephritis were statistically significantly decr-eased in their proliferative activities , apoptosis and CD28 expression in a dose-dependent manner. No inhibitory effects on CD152 expression of T-lymphocytes had been observed . MSCs promoted the gene expression of TGF-β1 and inhibited the gene expression of IL-10, IFN-γ. Conclusion MSCs can inhibit the proliferative activities, apoptosis and CD28 expression of peripheral blood T-lymphocytes of lupus nephritis, increase gene expression of TGF-β1 and lower the gene expression of IL-10, IFN-γ, which may play an important role in it's immunosuppressive effects on lupus nephritis.