Effects of small GTP-binding protein GDP dissociation stimulator on adipocyte hypertrophy and glucose metabolism disorder in mice / 中华内科杂志

Objective: To explore the effect and mechanism of small GTP-binding protein GDP dissociation stimulator (SmgGDS) on the development of obesity. Methods: (1) 8-week-old C57BL/6J mice were randomly assigned to normal diet and high fat diet group, with 6 mice in each group. They were fed regular feed and a high fat diet containing 60% fat for 4 months, respectively. The expression of SmgGDS in epididymal adipose tissue (eWAT), liver, and skeletal muscle were measured using Western-blot. (2) 6-week-old wild-type (WT) and SmgGDS knockdown (KD) mice were divided into four groups, each receiving high fat diet for 4 months (7 in each group) and 7 months (9 in each group). Glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted; The weight, adipose tissue, and liver weight of mice were recorded; HE staining examined adipose tissue structural changes; Western-blot determined extracellular signal-regulated kinase (ERK) 1/2 phosphorylation levels in eWAT; Real time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA levels of CCAAT/enhancer binding protein α (C/EBPα), C/EBPβ and peroxisome proliferator activated receptor γ (PPARγ) in eWAT. (3) Mouse embryonic fibroblasts (MEFs) extracted from WT and KD mice were induced for differentiation. Oil red O staining and Western-blot were used to detect lipid droplet and expression of SmgGDS and phospho-ERK; C/EBPα, C/EBPβ and PPARγ mRNA levels were measured using RT-qPCR. (4) 10-week-old C57BL/6J mice were randomly assigned into two groups, with 7 mice in each group. Mice were infected with SmgGDS overexpressing adeno-associated virus (AAV-SmgGDS) or empty vector intraperitoneally, then fed with high fat diet. After 4 weeks, performed GTT and ITT; Recorded the weight and adipose tissue weight of mice; HE staining was used to analyze structural changes of eWAT; Western-blot was used to detect the phosphorylation level of ERK in eWAT. Results: (1) The expression of SmgGDS was significantly upregulated in eWAT of high fat diet fed mice (normal diet group: 0.218±0.037, high fat diet group:0.439±0.072, t=2.74, P=0.034). (2) At 4 months of high fat diet intervention, the glucose tolerance (60 minutes after glucose injection, WT group: 528 mg/dl±21 mg/dl, KD group: 435 mg/dl±17 mg/dl, t=3.47, P=0.030; 90 minutes, WT group: 463 mg/dl±24 mg/dl, KD group: 366 mg/dl±18 mg/dl, t=3.23, P=0.047;120 minutes, WT group: 416 mg/dl±21 mg/dl, KD group: 297 mg/dl±16 mg/dl, t=4.49, P=0.005) and insulin sensitivity (15 minutes after insulin injection, WT group: 77.79%±3.45%, KD group: 54.30%±2.92%, t=3.49, P=0.005; 30 minutes, WT group: 62.27%±5.31%, KD group: 42.25%±1.85%, t=2.978, P=0.024; 90 minutes, WT group: 85.69%±6.63%, KD group: 64.71%±5.41%, t=3.120, P=0.016) of KD mice were significantly improved compared to the WT group, with an increase in eWAT weight ratio (WT: 4.19%±0.18%, KD: 5.12%±0.37%, t=2.28, P=0.042), but a decrease in average adipocyte area (WT group: 5221 μm²±241 μm², KD group: 4410 μm²±196 μm², t=2.61, P=0.026). After 7 months of high fat diet, the eWAT weight ratio of KD mice decreased (WT: 5.02%±0.20%, KD: 3.88%±0.21%, t=3.92, P=0.001) and adipocyte size decreased (WT group: 6 783 μm²±390 μm², KD group: 4785 μm²±303 μm², t=4.05, P=0.002). The phospho-ERK1 in eWAT increased (WT group: 0.174±0.056, KD group: 0.588±0.147, t=2.64, P=0.025), and mRNA level of PPARγ significantly decreased (WT group: 1.018±0.128, KD group: 0.029±0.015, t=7.70, P=0.015). (3) The expression of SmgGDS was significantly increased in differentiated MEF (undifferentiated: 6.789±0.511, differentiated: 10.170±0.523, t=4.63, P=0.010); SmgGDS knock-down inhibited lipid droplet formation in MEF (WT group: 1.00±0.02, KD group: 0.88±0.02, t=5.05, P=0.007) and increased ERK1 (WT group: 0.600±0.179, KD group: 1.325±0.102, t=3.52, P=0.025) and ERK2 (WT group: 2.179±0.687, KD group: 5.200±0.814, t=2.84, P=0.047) activity, which can be reversed by ERK1/2 inhibitor. (4) SmgGDS over expression resulted in weight gain, increased eWAT weight (control group: 3.29%±0.36%, AAV-SmgGDS group: 4.27%±0.26%, t=2.20, P=0.048) and adipocyte size (control group: 3525 μm²±454 μm², AAV-SmgGDS group: 5326 μm²±655 μm², t=2.26, P=0.047), impaired insulin sensitivity(30 minutes after insulin injection, control group: 44.03%±4.29%, AAV-SmgGDS group: 62.70%±2.81%, t=3.06, P=0.019), and decreased ERK1 (control group: 0.829±0.077, AAV-SmgGDS group: 0.326±0.036, t=5.96, P=0.001)and ERK2 (control group: 5.748±0.287, AAV-SmgGDS group: 2.999±0.845, t=3.08, P=0.022) activity in eWAT. Conclusion: SmgGDS knockdown improves obesity related glucose metabolism disorder by inhibiting adipogenesis and adipose tissue hypertrophy, which is associated with ERK activation.

Blockage of allergic rhinitis in mice with recombinant protein of cytotoxic T lymphocyte associated antigen-4 extracellular domain / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To explore a new immunotherapy against allergic rhinitis.</p><p><b>METHODS</b>The recombinant protein of CTLA4 extracellular domain was obtained through construction of CTLA4-yeast expression system. The allergic rhinitis in mice was induced by sensitizing and challenging with ovalbumin (OVA). The allergic rhinitis related symptoms and the morphological changes in nasal mucosa were compared between the allergic rhinitis group and the CTLA4 extracellular domain group treated with CTLA4 extracellular domain before each challenge by ways of intraperitoneal injection.</p><p><b>RESULTS</b>CTLA4 extracellular domain with a molecular weight of 28 000, which was confirmed by Western blot, could be generated through CTLA4-yeast expression system. The purified CTLA4 extracellular domain could inhibit T cells proliferation in mixed lymphocyte reaction with a inhibitory rate of 95.4%. The mice in allergic rhinitis group appeared typical allergic rhinitis symptoms after OVA challenge, such as rhinorrhea and sneeze. Meanwhile the nasal pathological studies showed edema and congestion in mucosa tissue and local influx of inflammatory cells. Whereas in CTLA4 extracellular domain group, the nasal symptoms were rarely observed, and the pathological change in nasal mucosa was significantly abated.</p><p><b>CONCLUSIONS</b>The protein of CTLA4 extracellular domain could prevent the allergic rhinitis in mice. The underlying mechanism of which might be the inhibition of the T cell activation.</p>

Experimental study on the antibacterial effects of zirconium phosphate gauze loaded with silver on the common bacteria on rat burn wound / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the antibacterial effects of zirconium phosphate gauze loaded with silver on rat burn wounds seeded with commonly seen bacteria.</p><p><b>METHODS</b>Wistar rats were employed in the study and were scalded and infected. The minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC) of zirconium phosphate loaded with silver were determined by double dilution in tubes. The effect on wound healing and the subeschar bacterial count of the rat burn wounds were observed after the wounds had been covered by gauze loaded with zirconium phosphate and silver, and also with the gauze which has been rinsed for 20 times.</p><p><b>RESULTS</b>The MIC of silver loaded zirconium phosphate on Staphylococcus aureus, Pseudomonas aeruginosa and E. coli were 8, 8 and 16 mg/L, respectively, while the MBC were 16, 8 and 32 mg/L, respectively. The subeschar bacterial count in the burn wounds with the gauze with silver loaded zirconium phosphate was ten times lower than that in those which were treated with gauze with SD-Ag and 100 times lower than that with ordinary gauze. But there was no difference in the bacterial count between the wounds which were treated with fresh gauze with silver loaded zirconium phosphate and that with the gauze which has been rinsed for 20 times (P > 0.05). Furthermore, wound healing seemed to be better with the gauze with silver loaded zirconium phosphate when compared with those by the other two kinds of gauze.</p><p><b>CONCLUSION</b>The silver loaded zirconium phosphate was found to be bacteriocidal against bacteria commonly seen in the burn wounds.</p>

The influence of the application of cytotoxic lymphocyte antigen 4-Ig adenovirus on the burn wounds with alloskin grafting on the murine immune function / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of local application of cytotoxic lymphocyte antigen 4-Ig (CTLA4-Ig) adenovirus on the burn wound with alloskin grafting upon the murine immune function.</p><p><b>METHODS</b>Sixty BALB/c mice were randomly divided into A (operation control), B (CTLA4-Ig transfection) and C (normal control) groups, with 20 mice in each group. Skin wounds (full-thickness loss) sized 1.5 cm x 1.5 cm were created on the backs of mice in A and B groups. Then the skin grafts of the same size obtained from C57BL mice were grafted into the skin wounds. 0.1 g of cross-linking polyacrylic resin (carbomer cream) without adenovirus was daubed onto the wounds in A group, and the same amount of carbomer cream with adenovirus in titers of 5 x 10(9)/L was daubed onto the wounds in B group, while no treatment was given in C group. 1 ml of 10% SRBC (sheep red blood cell) was injected intraperitoneally to all the mice of the three groups on the 1st post injury day (PID). Splenocytes from BALB/c, C57BL and Kunming mice were harvested for mixed lymphocyte culture on 7, 14, 21 and 28 PIDs. Agglutination assay was used in the same time to detect the SRBC antibody titers.</p><p><b>RESULTS</b>The reaction of murine splenocytes in B group to the donor (C57BL) splenocytes was suppressed in a specific way (P < 0.05) within 14 PIDs. There was no difference in the titers of anti-SRBC antibody among the 3 groups (P > 0.05).</p><p><b>CONCLUSION</b>Local application of CTLA4-Ig recombinant adenovirus exhibited no influence on the murine humoral immunity, but might induce systemic and specific T cell tolerance in immunity system.</p>

An experimental study on the role of indirect antigen presentation pathway on the immunogenetic of epidermal cells / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the role of indirect antigen presentation pathway on the immunogenecity of epidermal cells.</p><p><b>METHODS</b>Human epidermal cells (HEC), allogeneic human peripheral blood lymphocytes (PBL) and mononuclear cells (PBM, including monocytes) were isolated and cultured in vitro. HECs were transfected by human-originated CTLA4Ig-adenovirus vector. The CTLA4Ig expression was observed. Allogeneic PBLs or PBMs were added to the transfected and non-transfected HECs with simple cultured PBLs and PBMs as the control. The proliferation of PBL and PBM was determined by (3)H-TdR incooperation.</p><p><b>RESULTS</b>HECs could be successfully transfected by CTLA4Ig-adenovirus vector and expressed corresponding proteins. The non-transfected HECs could stimulate slight proliferation of allogeneic PBLs (P < 0.05) and stimulate remarkable proliferation of PBMs (including monocytes) (P < 0.05). The proliferation reaction of PBLs and PBMs decreased significantly (P < 0.05) after being stimulated by HEC which was modulated by CTLA4Ig genes.</p><p><b>CONCLUSION</b>Indirect antigen presentation pathway might play important roles in the HEC immunogenicity which could be evidently inhibited by CTLA4Ig.</p>