Objective The purpose of this study was to provide a more effective method for researching the prevention and treatment of Graves'disease by comparing the effects of two plasmid vectors expressing the human thyrotropin receptor(TSHR)A subunit gene in inducing an animal model of Graves'disease via electroporation.Methods Plasmids pcDNA3.1-THSR A,and pTriEx1.1-THSR A expressing the TSHR A subunit were constructed and used to induce Graves'disease by intramuscular injection with immediate electroporation once every 3 weeks for a total of 4 times.Mice in the control group were injected with PBS.One week after the second electroporation,blood was collected to measure serum thyrotropin receptor antibody(TRAb).Three weeks following the last electroporation,echocardiography was performed on the mice.Mice were sacrificed 4 weeks after the last electroporation;blood,thyroid,and orbital tissues were collected;serum total thyroxine(TT4)was measured;and histological examination was performed.Results The average concentrations of serum TRAb in the pcDNA3.1-TSHR A group(n=15)and the pTriEx1.1-TSHR A group(n=13)were(6.9±2.0)U/L and(7.5±2.2)U/L,respectively.The latter was significantly higher than that in the control group(4.9±0.5)U/L(P=0.033).The average concentrations of serum TT4 in the pcDNA3.1-TSHR A group and pTriEx1.1-TSHR A group were(41.4±23.8)ng/mL and(63.2±53.7)ng/mL,respectively,both higher than that in the control group:(20.2±4.0)ng/mL(P＜0.01).Thyroid pathology showed thyroid follicular epithelial hyperplasia with T-cell infiltration in the model group.Echocardiography showed that the left ventricle mass in the pTriEx1.1-TSHR A group was higher than those in the control group(P=0.007)and pcDNA3.1-TSHR A group(P=0.012).Orbital pathology showed fibrotic changes in the extraocular muscles of mice in the model groups.Conclusions Both pcDNA3.1 and pTriEx1.1 expressing the TSHR A subunit were able to induce Graves'disease in mice by electroporation,and the efficiency of the two plasmids in inducing hyperthyroidism and Graves'ophthalmopathy was similar.The efficiency of pTriEx 1.1-TSHR A in inducing thyrotoxic heart disease was better than that of pcDNA3.1-TSHR A.

Objective The purpose of this study was to provide a more effective method for researching the prevention and treatment of Graves'disease by comparing the effects of two plasmid vectors expressing the human thyrotropin receptor(TSHR)A subunit gene in inducing an animal model of Graves'disease via electroporation.Methods Plasmids pcDNA3.1-THSR A,and pTriEx1.1-THSR A expressing the TSHR A subunit were constructed and used to induce Graves'disease by intramuscular injection with immediate electroporation once every 3 weeks for a total of 4 times.Mice in the control group were injected with PBS.One week after the second electroporation,blood was collected to measure serum thyrotropin receptor antibody(TRAb).Three weeks following the last electroporation,echocardiography was performed on the mice.Mice were sacrificed 4 weeks after the last electroporation;blood,thyroid,and orbital tissues were collected;serum total thyroxine(TT4)was measured;and histological examination was performed.Results The average concentrations of serum TRAb in the pcDNA3.1-TSHR A group(n=15)and the pTriEx1.1-TSHR A group(n=13)were(6.9±2.0)U/L and(7.5±2.2)U/L,respectively.The latter was significantly higher than that in the control group(4.9±0.5)U/L(P=0.033).The average concentrations of serum TT4 in the pcDNA3.1-TSHR A group and pTriEx1.1-TSHR A group were(41.4±23.8)ng/mL and(63.2±53.7)ng/mL,respectively,both higher than that in the control group:(20.2±4.0)ng/mL(P＜0.01).Thyroid pathology showed thyroid follicular epithelial hyperplasia with T-cell infiltration in the model group.Echocardiography showed that the left ventricle mass in the pTriEx1.1-TSHR A group was higher than those in the control group(P=0.007)and pcDNA3.1-TSHR A group(P=0.012).Orbital pathology showed fibrotic changes in the extraocular muscles of mice in the model groups.Conclusions Both pcDNA3.1 and pTriEx1.1 expressing the TSHR A subunit were able to induce Graves'disease in mice by electroporation,and the efficiency of the two plasmids in inducing hyperthyroidism and Graves'ophthalmopathy was similar.The efficiency of pTriEx 1.1-TSHR A in inducing thyrotoxic heart disease was better than that of pcDNA3.1-TSHR A.

Objective:To evaluate the effect of sodium sivelestat on the expression of specialized pro-resolving mediators (SPMs) synthesis enzymes in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI).Methods:Eighteen SPF healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 3 groups ( n=6 each) using a random number table method: control group (C group), LPS-induced ALI group (ALI group), and LPS-induced ALI + sodium sivelestat group (ALI+ SV group). ALI was induced by intravenous injection of LPS 15 mg/kg through the tail vein. Sodium sivelestat 50 mg/kg was intraperitoneally injected at 1 h after LPS administration. At 12 h after LPS administration, blood samples were collected from the eyeballs for routine blood tests, and the remaining blood was processed for serum extraction. The mice were sacrificed after anesthesia, and lung tissues were collected to determine the wet/dry weight (W/D) ratio, serum concentrations of interleukin-1beta (IL-1β) and IL-10 (by enzyme-linked immunosorbent assay), expression of neutrophil elastase (NE) and SPMs synthesis enzymes 5-lipoxygenase (5-LOX), 12-lipoxygenase (12-LOX), and 15-lipoxygenase (15-LOX) in lung tissues (by Western blot) and to examine the pathological changes of lung tissues which were scored. Results:Compared with C group, the lung injury scores, W/D ratio, white blood cell counts, percentage of neutrophil, and serum IL-1β and IL-10 concentrations were significantly increased, the expression of NE was up-regulated, and the expression of 5-LOX, 12-LOX and 15-LOX was down-regulated in ALI group ( P<0.05). Compared with ALI group, the lung injury scores, W/D ratio, white blood cell counts, percentage of neutrophil, and serum IL-1β concentration were significantly decreased, the serum IL-10 concentration was increased, the expression of NE was down-regulated, and the expression of 5-LOX, 12-LOX and 15-LOX was up-regulated in ALI+ SV group ( P<0.05). Conclusions:The mechanism by which sodium sivelestat alleviates LPS-induced ALI may be related to up-regulating the expression of SPMs synthesis enzyme and promoting the resolution of pulmonary inflammation in mice.

Objective:To investigate the effect of nuclear factor E2-related factor 2 (Nrf2) on the cellular tight junction protein Claudin-18 in endotoxin-induced acute lung injury (ALI).Methods:Eighteen healthy male C57BL/6 mice were divided into control group, endotoxin-induced ALI model group (ALI group) and Nrf2 activator tert-butylhydroquinone (tBHQ) pretreatment group (tBHQ+ALI group) according to random number table method, with 6 mice in each group. Mice endotoxin-induced ALI model was reproduced by intraperitoneal injection of lipopolysaccharide (LPS, 15 mg/kg), and the mice in the control group was injected with an equal amount of phosphate buffer solution (PBS). The mice in the tBHQ+ALI group received three intraperitoneal injections of tBHQ (a total of 50 mg/kg) at an interval of 1 hour before molding. The last injection of tBHQ was accompanied by LPS of 15 mg/kg. The mice in the control group and model group were given equal amounts of PBS, and PBS or LPS was given at the last injection. The mice were sacrificed at 12 hours after LPS injection to take lung tissues. After the lung tissue was stained with hematoxylin-eosin (HE) staining, the pathological changes were observed under light microscopy, and the lung injury score was calculated. The lung wet/dry ratio (W/D) was determined. Nrf2 protein expression in the lung tissue was detected by Western blotting. Positive expression of Claudin-18 in the lung tissue was determined by immunohistochemistry.Results:The lung tissue showed normal structure, without significant pathological change in the control group. Compared with the control group, the alveolar septum widened accompanied by inflammatory cell infiltration, capillary hyperemia and tissue edema in the ALI group, the lung injury score and lung W/D ratio were significantly increased (lung injury score: 6.50±1.05 vs. 1.83±0.75, lung W/D ratio: 3.79±0.22 vs. 3.20±0.14, both P < 0.01), and the Nrf2 protein expression and Claudin-18 positive expression in the lung tissue were significantly lowered [Nrf2 protein (Nrf2/β-actin): 0.41±0.33 vs. 1.22±0.33, Claudin-18 ( A value): 0.28±0.07 vs. 0.44±0.10, both P < 0.05]. After tBHQ pretreatment, the degree of lung histopathological injury was significantly reduced compared with the ALI group, the alveolar space slightly abnormal, inflammatory cell infiltration and tissue edema reduced, the lung injury score and lung W/D ratio were significantly decreased (lung injury score: 3.00±0.89 vs. 6.50±1.05, lung W/D ratio: 3.28±0.19 vs. 3.79±0.22, both P < 0.01), and Nrf2 protein expression and Claudin-18 positive expression in the lung tissue were significantly increased [Nrf2 protein (Nrf2/β-actin): 1.26±0.09 vs. 0.41±0.33, Claudin-18 ( A valure): 0.45±0.04 vs. 0.28±0.07, both P < 0.05]. Conclusion:Nrf2 alleviated pulmonary edema and improved endotoxin-induced ALI by up-regulating Claudin-18 expression.

Objective:To evaluate the role of heme oxygenase-1 (HO-1) in endotoxin-induced acute lung injury (ALI) and the relationship with the regulation of mitochondrial quality control in mice.Methods:Clean-grade healthy male adult C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were selected.HO-1 inducible gene knockout mice (HO-1 -/-) were prepared based on CRISPER/Cas9-mediated EGE system, and HO-1 gene overexpression mice (HO-1 + /+ ) were prepared by transfection of HO-1 overexpressed adenovirus vector.The mice were divided into 2 groups ( n=6 each) using a random number table method: control group (group WT, group HO-1 -/-, group HO-1 + /+ ) and endotoxin-induced ALI group (group ALI, group HO-1 -/-+ ALI, group HO-1 + /+ + ALI). Lipopolysaccharide 15 mg/kg was injected through the tail vein to develop the model of endotoxin-induced ALI, and the equal volume of normal saline was given instead in each control group.The mice were sacrificed by bloodletting at 12 h after lipopolysaccharide or normal saline administration.The lung tissues were harvested for microscopic examination of the pathological changes which were scored, for determination of GSH and GSSG contents, for observation of the ultrastructure of mitochondria (with a transmission electron microscope) and survival within 12 h, for measurement of mitochondrial membrane potential (MMP) levels, and for determination of the expression of mitochondrial quality control-related proteins mitochondrial fusion protein 2 (Mfn2) and dynamin-related protein 1 (Drp1), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitophagy marker protein PTEN-induced kinase 1 (PINK1) and E3 ubiquitin-protein ligase Parkin.The ratio of GSH/GSSG was calculated. Results:Compared with control group (group WT, group HO-1 + /+ and group HO-1 -/-), the 12-h survival rate and MMP were significantly decreased, the lung injury score was increased, GSH content and GSH/GSSG ratio were decreased, and the content of GSSG was increased in endotoxin-induced ALI groups (group ALI, group HO-1 + /+ + ALI and group HO-1 -/-+ ALI) ( P<0.05). Compared with group ALI, the 12-h survival rate and MMP were significantly decreased, the lung injury score was increased, the GSH content and GSH/GSSG ratio were decreased, the GSSG content was increased, and the expression of HO-1, Mfn2, PGC-1α, NRF1, PINK1 and Parkin was down-regulated, and Drp1 expression was up-regulated in group HO-1 -/-+ ALI, and 12-h survival rate and MMP were significantly increased, lung injury score was decreased, GSH content and GSH/GSSG ratio were increased, GSSG content was decreased, the expression of HO-1, Mfn2, PGC-1α, NRF1, PINK1 and Parkin was up-regulated, and the expression of Drp1 was down-regulated in group HO-1 + /+ + ALI ( P<0.05). Conclusions:HO-1 is involved in the process of endotoxin-induced ALI in mice, which is related to the regulation of mitochondrial quality control.

Objective:To evaluate the effect of transcutaneous electrical acupoint stimulation (TEAS) on mitochondrial quality control during endotoxin-induced acute lung injury (ALI) in mice.Methods:Twenty-four clean-grade healthy male C57BL/6J mice, aged 4-6 weeks, weighing 15-20 g, were divided into 4 groups ( n=6 each) according to the random number table method: control group (group C), endotoxin-induced ALI group (group L-ALI), endotoxin-induced ALI plus acupoint electroacupuncture group (group L-ALI+ EA), and endotoxin-induced ALI plus non-acupoint electroacupuncture group (group L-ALI+ SEA). Lipopolysaccharide (LPS) 15 mg/kg was injected via the caudal vein to develop the model of endotoxin-induced ALI in anesthetized mice.In group L-ALI+ EA, at 5 days before LPS injection, bilateral Zusanli and Feishu acupoints were stimulated with an electric stimulator for 30 min each time at a voltage of 1-2 mA and a frequency of 2/15 Hz until the end of the experiment.In group L-ALI+ SEA, stimulation was performed at the points 0.5 cm lateral to the acupoints of bilateral Zusanli and Feishu non-meridian and non-acupoint sites using the shallow puncture method, and the other treatment methods were the same as those previously described in group EA.Group C received no treatment.The mice were sacrificed by euthanasia at 12 h after LPS administration, and lung tissues were obtained for microscopic examination of the pathological changes (with a light microscope) and structure and morphology of mitochondria (with a transmission electron microscope) and for determination of the levels of reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) and contents of glutathione (GSH) and glutathione oxidized (GSSG). The GSH/GSSG ratio was calculated.The expression of mitochondrial fusion proteins mitofusin 1 (Mfn1), Mfn2, optic atrophy1 (OPA1), dynamin-related protein 1 (Drp1), fission protein 1 (Fis1), peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor-1 (NRF1), NRF2, PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase (Parkin) was determined by Western blot. Results:Compared with group C, the level of ROS and contents of GSSG and mtDNA were significantly increased, GSH content and GSH/GSSG ratio were decreased, the expression of Mfn1, Mfn2, OPA1, NRF1, NRF2 and PGC-1α was down-regulated, and the expression of Drp1, Fis1, PINK1 and Parkin was up-regulated in L-ALI, L-ALI+ EA and L-ALI+ SEA groups ( P<0.05). Compared with group L-ALI, the level of ROS and contents of GSSG and mtDNA were significantly decreased, GSH content and GSH/GSSG ratio were increased, the expression of Mfn1, Mfn2, OPA1, NRF1, NRF2 and PGC-1α was up-regulated, and the expression of Drp1, Fis1, PINK1 and Parkin was down-regulated in group L-ALI+ EA ( P<0.05). Compared with group L-ALI+ EA, the level of ROS and contents of GSSG and mtDNA were significantly increased, GSH content and GSH/GSSG ratio were decreased, the expression of Mfn1, Mfn2, OPA1, NRF1, NRF2 and PGC-1α was down-regulated, and the expression of Drp1, Fis1, PINK1 and Parkin was up-regulated in group L-ALI+ SEA ( P<0.05). Conclusions:TEAS can reduce endotoxin-induced ALI probably through regulating mitochondrial quality control in mice.

Objective To discuss the methods and clinical effects of phased treatment of ankylosing spondylitis (AS) combined with severe hip flexion contracture.Methods The study enrolled 8 cases (12 hips) of AS combined with severe hip flexion contracture hospitalized from September 2011 to November 2012.Phased treatments included lateral hip arthrolysis,articular capsulectomy,stripping of the reflected head of rectus femoris,femoral neck osteotomy,traction and stage Ⅱ biotype total hip arthroplasty (THA).Preoperative and postoperative Harris score,hip range of motion,and complication of femoral nerve injury were detected.Results Period of follow-up was 6-12 months (mean 10 months).One case developed heterotopic ossification,which affected postoperative hip activity and received resection one year later.One case sustained fissure fracture of calcar femorale during implantation of the prosthetic femoral stem,but no special handling was provided.Of all cases,active flexion and extension of the hip were both 0° before operation,but increased to (96.25 ± 4.33) ° and (24.17 ± 4.69)° respectively after operation ; mean Harris score was improved from (26.67 ± 2.39) points preoperatively to (90.92 ± 5.66) points postoperatively (P ＜ 0.01).Besides,no femoral nerve injury was observed.Conclusion Phased treatment of AS combined with severe hip flexion contracture restores hip function and minimizes femoral nerve injury following THA.