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Objective:To evaluate the role of sonic hedgehog (Shh)/glioma-associated oncogene homolog 1 (Gli1) signaling pathway in sleep deprivation-induced cognitive impairment in young mice.Methods:Forty-eight SPF healthy male C57BL/6 mice, aged 4 weeks, weighing 14-16 g, were divided into 3 groups ( n=16 each) by the random number table method: control group (C group), sleep deprivation group (SD group) and Shh agonist SAG group (SD+ SAG group). Multi-platform water environment method was used to prepare the sleep deprivation model in mice, and the sleep deprivation was 20 h a day for 10 consecutive days.In SD+ SAG group, SAG 10 mg/kg was intraperitoneally injected at 5 min before each sleep deprivation, while the equal volume of normal saline was intraperitoneally injected in group C and group SD.The mice underwent novel object recognition and Y-maze tests at 24 h after development of the model.Mice were sacrificed after the behavioral testing, and the hippocampi were isolated for determination of the density of dendritic spines in hippocampal CA1 region (by Golgi staining), expression of Gli1 and brain-derived neurotrophic factor (BDNF) in hippocampal tissues (by Western blot), and expression of Gli1 and BDNF mRNA in hippocampal tissues (by quantitative real-time polymerase chain reaction). Results:Compared with group C, the preference index in novel object recognition and Y-maze tests and density of dendritic spines in CA1 region were significantly decreased, and the expression of Gli1 and BDNF protein and mRNA in hippocampus was down-regulated in group SD ( P<0.05). Compared with group SD, the preference index in novel object recognition and Y-maze tests and density of dendritic spines in CA1 region were significantly increased, and the expression of Gli1 and BDNF protein and mRNA in hippocampus was up-regulated in group SD+ SAG ( P<0.05). Conclusions:Inhibition of Shh/Gli1 signaling pathway and reduction of plasticity of dendritic spines of hippocampal neurons are involved in sleep deprivation-induced cognitive impairment in young mice.
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Objective To evaluate the relationship between mechanical ventilation-induced apoptosis in hippocampal neurons and mammalian taget of rapamycin ( mTOR) signaling pathway in mice. Methods Fifty healthy male C57BL∕6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 2 groups ( n=25 each) using a random number table method: control group ( group C ) and mechanical ventilation group ( group V) . The mice breathed spontaneously for 6 h in group C, and the mice were mechanically ventilated for 6 h in group V. Open field test and contextual fear conditioning test were conducted at 1 and 3 days after the end of ventilation. Hippocampal tissues were obtained at 1 day after the end of ventilation for determina-tion of the expression of mTOR, phosphorylated mTOR (p-mTOR), microtubule-associated protein 1 light chain 3Ⅱ( by Western blot) and apoptosis in hippocampal neurons ( by TUNEL) . The p-mTOR∕mTOR ratio and apoptosis index were calculated. Results Compared with group C, the time animals spent in the central square was significantly prolonged, the number of crossing the grid was reduced, the percentage of freezing time was decreased, the expression of microtubule-associated protein 1 light chain 3Ⅱwas up-regulated, and the p-mTOR∕mTOR ratio and apoptosis index were increased in group V ( P<0. 05) . Conclusion The mech-anism by which mechanical ventilation induces apoptosis in hippocampal neurons may be related to activation of mTOR signaling pathway in mice.
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@#Objective To explore the effects of lycopene on interleukin-6 (IL-6) expression and motor function after spinal cord injury in rats. Methods 36 healthy adult Sprague-Dawley rats were randomly divided into control group (A), methylprednisolone sodium succinate (MP) treatment group (B) and lycopene treatment group (C) with 12 rats in each group, and spinal cord injury model at T9 was established with modified Allen's technique (10 g×25 mm). 30 minutes after modeling, group A received no treatment, group B was injected MP 30 mg/ kg, group C was given lycopene 20 mg/kg. They were tested with inclined plate test 1 day, 3 days and 7 days after injury. The expression of IL-6 was examined with immunohistochemistry, then. Results The inclined plate test scores were higher in the group C than in the group A 1 day and 7 days after injury (P<0.05), and in the group B than in the group A 1 day, 3 days and 7 days after injury (P<0.05). The expression of IL-6 was significantly lower in the groups B and C than in the group A 1 day, 3 days, 7 days after injury (P<0.001). Conclusion Lycopene can inhibit the expression of IL-6 in acute spinal cord injury to reduce the inflammation and facilitate the recovery of nerve and motor function.
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Objective To explore the effects of lycopene on interleukin-6 (IL-6) expression and motor function after spinal cord injury in rats. Methods 36 healthy adult Sprague-Dawley rats were randomly divided into control group (A), methylprednisolone sodium succinate (MP) treatment group (B) and lycopene treatment group (C) with 12 rats in each group, and spinal cord injury model at T9 was established with modified Allen's technique (10 g×25 mm). 30 minutes after modeling, group A received no treatment, group B was injected MP 30 mg/kg, group C was given lycopene 20 mg/kg. They were tested with inclined plate test 1 day, 3 days and 7 days after injury. The expression of IL-6 was examined with immunohistochemistry, then. Results The inclined plate test scores were higher in the group C than in the group A 1 day and 7 days after injury (P<0.05), and in the group B than in the group A 1 day, 3 days and 7 days after injury (P<0.05). The expression of IL-6 was significantly lower in the groups B and C than in the group A 1 day, 3 days, 7 days after injury (P<0.001). Conclusion Lycopene can inhibit the expression of IL-6 in acute spinal cord injury to reduce the inflammation and facilitate the recovery of nerve and motor func-tion.
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Objective:To investigate the expression and mechanism of NF-κB signal pathway in murine lupus nephritis.Methods:The BXSB mice as well as C57BL/6 of 16 weeks were used.Transmission electron microscope and PAS were used to detect the pathological change of renal tissue.RT-PCR and ELISA were used to detect the expression of HMGB1 mRNA and protein.The expression of HMGB1,p- NF-κB,RAGE,IκB and PCNA protein was detected by immunohistochemical stain,FCM and Western blot.Results:The level of BUN in serum and Micro-albumin in urine of BXSB mice was higher than that in C57BL/6 mice.The expression of HMGB1 mRNA and HMGB1 protein level in peripheral blood increased significantly in BXSB group.Compared with those in control group,electron microscopy and PAS revealed the thickness of glomerular basement membrane(GBM),fusion of foot processes partly of epithelial dell and subepithelial electron-dense deposits in the renal tissue of BXSBA mice.Compared with that of control group,expression of PCNA was higher in glomeruli of BXSB mouse.HMGB1 protein over-expression localized in cytoplasm and extracellular milieu,especially in proliferative glomeruli in BXSB group,while the HMGB1 protein primarily confined to the nuclear of tubule in control group.In BXSB group,the expression of p-NF-κB and RAGE increased,while the expression of IκB decreased.There were positive correlation between the expression of HMGB1,RAGE and p-NF-κB protein (r=0.833,0.621,0.848,P＜0.01),while the expression of p-NF-κB protein negatively correlated with that of IκB.Conclusion:HMGB1 could activate NF-κB through combining with its receptor-RAGE,induce the form of proliferative glomerulonephritis by promoting the proliferation of inherent cell of glomeruli,which may play an important role in the murine lupus nephritis.
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Objective To investigate the effect and possible mechanism of high mobility group box (HMGB) 1 in the development and progress of rheumatoid arthritis.Methods PBMC and serum samples were obtained from 74 RA patients (38 in active stage and 36 in stable stage) and 26 healthy controls.The expression of HMGB1 mRNA and protein was detected by RT-PCR and ELISA.Flow cytometry analysis ( FCM ) was used to detect the expression of Toll-like receptor 4 on PBMC.Results ①The expression of HMGBI mRNA and protein in active RA patients was significantly higher than that in healthy controls and inactive RA patients [2.63 vs 0.71，0.93 and (10.2±1.2) vs (7.5±1.8)，(8.3±1.8) ng/ml，respectively](P＜0.01 ).② The relative expression of TLR4 protein on CD14+ monocytes and CD3+ lymphocytes in active RA patients was increased than that in inactive RA and healthy controls (P＜0.05 or P＜0.01 ).It was also higher in inactive RA than in healthy controls (P＜0.05 or P＜0.01 ).③ Level of HMGB1 protein in serum of RA patients was positively correlated with ESR，CRP，RF，the numbers of tender joints and swollen joints as well as radiographic changes.Conclusion HMGB1 can be synthesized and released by PBMC of active RA patients，and then bind to TLR4 of PBMC to promote inflammatory responses and bone erosion.
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Objective To investigate the relationship between the effect of high mobility group protein box 1 (HMGBI) on the renal injure of systemic lupus erythematosus (SLE) and the expression of Toll-like receptor 4(TLR4). Methods The level of HMGB1, MMP-2 and TIMP-2 in serum from 16 pa-tients with SLE, 18 patients with lupus nephritis(LN) and 12 healthy people were measured by ELISA. The fresh peripheral blood mononuelear cell (PBMC) were isolated and the total RNA was extracted. Then the mRNA expression of HMGB1 was amplified by RT-PCR. Flow cytometry analysis was performed to study cell surface markers and the expression of TLR4. Results RT-PCR and ELISA results showed that the expres-sions of mRNA and level of HMGB1 protein in serum were higher in patients with LN than those in SLE and healthy people. The expression of TLR4 in CD14+ monecytes of patients with LN was higher than that with SLE and healthy people, while there were no significance in CD3+ T cells among LN, SLE and healthy peo-ple. The expressions of MMP-2 and TIMP-2 in serum of LN was lower than that in SLE and healthy people, at the same time the ratio of MMP-2/TIMP-2 decreased in LN group. HMGB1 mRNA and CD14+/TLR4+ was negatively correlated with the ratio of MMP-2/TIMP-2, and the level of HMGB1 in serum was positively correlated with proteinuria, while negatively correlated with the ratio of MMP-2/TIMP-2 in LN. Conclusion HMGB1 is one of the important cytokine in the pathogenesis of lupus nephritis. HMGBI might play a role in proteinuria of lupus nephritis in part via TLR4 pathway to activate monocytes and decrease the expression of MMP-2/TIMP-2.
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Objective To investigate the correlation between NF-?B/COX-2 signal pathway and cell proliferation in diabetic nephropathy. Methods Uninephrectomized STZ-induced male Wister rats were used as animal model. Using immunohistochemistry to detect NF-?B and COX-2 protein expressions in diabetic kidneys at the 16th week. HKC were cultured separately in normal or high glucose medium for 24,48,72 h.The expression of NF-?B and COX-2 protein was detected by flow cytometry and the expression of PCNA was detected by immunocytochemical staining. Results 1 Volum of glomeruli, mesangial matrix, thickness of glomerular and tubular basement membrane increased in diabete group; 2 COX-2 were expressed in cytoplasm of tubules and glomeruli by immunohistochemistry. Compared with control group, the expression of COX-2 was higher; activated NF-?B expressed in nucli of both tubules and glomeruli, There was light stainings for in control group, while enhanced stainings were observed in DM, there was a positive correlation between NF-?B and COX-2.3 Compared with those in HKC cultured in the medium with normal level glucose, the stainings were strengthened for PCNA in HKC exposed to high glucose from 24 h. 4 By FCM, the expression of NF-?B and COX-2 in HKC cultured in high glucose medium was higherthan that in normal glucose medium; the expression of NF-?B and PCNA was positively correlated with the expression of COX-2. Conclusion Activating NF-?B and elevating the expression of COX-2 play an important role in regulating cell proliferation, which may be one of the injury mechanisms of the renal cells during diabetic nephropathy.
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Objective:To investigate the effects of TLR/STAT pathway in the proliferation of mesangial cell induced by HMGB1.Methods:Human mesangial cells were inoculated in the dose of 1?104 ml-1.After 24 h,cells were cultured with standard medium as control group or with medium supplement with 10 ?g/L human recombinant protein HMGB1 as trial group in vitro.Then the cells were collected in 6,12 and 24 h respectively,as well as control group cells.Immunocytochemical staining was adopted to examine the expressions of PCNA proteins on mesangial cells in different groups.Immunocytochemical staining and FCM were performed to detect the changes of TLR2 protein expression.STAT1 and STAT3 mRNA were examined by RT-PCR technique.Results:Immunocytochemical staining indicated that the mesangial cells could multiply after they were induced by human recombinant protein HMGB1.Immunocytochemical staining showed that the level of TLR2 protein in trial groups were higher than those in control groups.FCM indicated that HMGB1 could significantly up-regulate the expression of TLR2 protein time-dependently.The STAT1 and STAT3 mRNA in HMGB1 groups were higher than those in control groups.The expression of TLR2 protein was positively correlated with those of STAT1 and STAT mRNA respectively.The positive rate of PCNA was remarkably correlated with the expression of STAT1 and STAT3 mRNA.Conclusion:HMGB1 could activate STAT1/STAT3 through combining with its cell-surface receptor TLR2,which may play an important role in promoting the proliferation of mesangial cells and then damaging the renal of lupus nephritis.
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Objective:To investigate the effect of HMGB1 and TLR-4 in the renal injury of SLE.Methods:The level of HMGB1 in serum from 16 patients suffering from SLE without kidney damage,18 patient with lupus nephritis (LN),and 12 healthy individuals were measured by ELISA.The fresh PBMCs were isolated and the total RNA was extracted.Then the mRNA expression of HMGB1 was amplified by RT-PCR.Flow cytometric analysis(FCM) was performed to study cell surface markers and the expression of TLR-4.Results:RT-PCR and ELISA results showed that the expressions of mRNA and protein were higher in patients with LN than in SLE without kindey damage and healthy people.The expression of TLR-4 in CD14+ monocytes of patients with LN was higher either,while there were no significant changes in CD3+ T cells among LN,SLE and healthy control.Conclusion:PBMCs in patients with LN could synthesize and secrete HMGB1 initiatively,which are correlated with serum HMGB1 level.HMGB1 might play a role in autoimmunity of lupus nephritis partly by activation of monocytes through its binding to TLR-4.