Background Studies showed that some members of matrix metalloproteinases (MMPs) play an important role in the pathogenesis of diabetic retinopathy (DR).However,whether MMPs inhibitor can deter blood retinal barrier (BRB) from damage is below understood.Objective This study was to investigate the effects of GM6001,a MMPs inhibitor,on BRB permeability.Methods Twenty-four adult clean SD rats were randomized to the control group,diabetic group and diabetes+GM6001 group according to randomized number table.Diabetic models were induced by the intraperitoneal injection of streptozotocin in the rats of the diabetic group and the diabetes + GM6001 group,and equal volume of citrate buffer was used in the same way in the rats of the control group.GM6001 10 μ1 (100 μ mol/L) was intravitreously injected in the third and fourteenth day after modeling in the diabetes+ GM6001 group,and equal volume of normal saline solution was injected in the same way in the control group and the diabetic group.The rats were sacrificed and eyeballs were extracted 1 month after injection,and the relative expressions of MMP-2 mRNA and MMP-9 mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR).Evens blue (EB) was infused via the right jugular vein,and paraformaldehyde solution 1％ was then infused via left ventricle at the perfusion pressure 120 mmHg.The eyeballs were extracted 2 minutes later,and the leakage of EB in rat retinas was examined.Results RT-PCR electrophoresis exhibited the response bands of MMP-2 mRNA,and MMP-9 mRNA and GAPDH,with the gene size of 436,536 and 484 bp,respectively.The difference of the MMP2 mRNA and MMP-9 mRNA was statisticaly significant (F =20.336,P =0.000 ; F =8.742,P =0.002) ; and the relative expressions of MMP-2 mRNA and MMP-9 mRNA were significantly higher in the diabetic group and diabetes +GM6001 group than those in the control group (all at P＜0.01),and the relative expressions of MMP-2 mRNA and MMP-9 mRNA in the diabetes+GM6001 group was significantly reduced in comparison with the diabetic group(both P=0.01,P=0.02).The standardized EB content in the retinas of the control group,diabetic group and diabetes+ GM6001 group was (12.60±3.50) ng/mg,(26.52±7.14) ng/mg and (17.55±2.65) ng/mg,showing a significant difference (F=17.032,P＜0.01),and EB content in rat retinas in the diabetic group was higher than that in the control group (P=0.003),and that in the diabetes+GM6001 group was lower in comparison with the diabetic group (P=0.020).Conclusions Intravitreal injection of GM6001 can down-regulate the expression of MMP-2 mRNA and MMP-9 mRNA in diabetic rats and therefore protect BRB.

Objective To observe the effect of panretinal photocoagulation (PRP) on the expression of cyclooxygenase-2 (COX-2),vascular endothelial cell growth factor (VEGF) in epiretinal membrane of proliferative diabetic retinopathy (PDR).Methods A total of 35 patients (35 eyes) with PDR and underwent plana vitrectomy were enrolled in this study.The patients were divided into non-PRP group (19 patients,19 eyes) and PRP group (16 patients,16 eyes) depends on if they had received PRP before surgery.The epiretinal membranes stripped during operation were collected for pathological examination.The histopathological features was observed by haematoxylin and eosin stain.The expression of CD34,COX-2 and VEGF,and microvessel density (MVD) were measured by immunohistochemistry method.Results Many new dispersed capillary blood vessels were found in the thick epiretinal membranes of nonPRP group,while scattered small blood vessels were found in the relatively thin epiretinal membranes of PRP group.MVD value was (7.42± 1.39) in the non-PRP group and (4.56± 1.22) in the PRP group,which was lower than the non-PRP group (t=6.41,P＜0.01).The expression of CD34,COX-2 and VEGF in the tissues of epiretinal membrane in PRP group were obviously lower than the non-PRP group (t=6.147,5.944,7.445;P＜0.01).Conclusion PRP can effectively inhibit the expression of COX-2 and VEGF in epiretinal membrane of PRP patients.

Objective To observe the effects of Astragalus polysaccharide ( APS) on tumor growth, cytokine and immune function impairment induced by cisplatin ( DDP) in mice bearing Lewis lung cancer.Methods A total of 90 mice were used in this study:10 for blank control group, and 80 mice with transplanted Lewis lung cancer were randomly divided into 8 groups:model control group (physiological saline), positive control group treated with DDP (6 mg/kg), low dose APS (50 mg/kg), moderate dose APS (100 mg/kg) and high dose APS (200 mg/kg) groups and three combinations of APS+DDP groups ( the same three APS levels with half dose of DDP, respectively) .0.3 mL of the drugs was intraper-itoneally injected to the mice, respectively, on the second day after moldeling.DDP was injected once a week and other drugs were injected once per day for consecutive 20 days.On the 21st day, blood samples were collected and serum levels of cytokine IL-2, IL-6, IL-12 and TNF-αwere determined by ELISA, and the tumor inhibition rate and immune organ in-dexes were assessed.Results The tumor inhibition rates of the positive control, low, moderate and high dose APS groups and three combinations of APS+DDP groups of mice bearing Lewis lung carcinoma were 49.30%, 17.21%, 39.68%, 17.21%, 51.02%, 57.21%and 65.11%, respectively.Compared with the model group, P<0.05 or P<0.01, and compared the three combination groups with the DDP group, P<0.05.Compared with the blank control group, the spleen index was significantly increased in the moderate and high dose APS groups and the three combinations of APS +DDP groups.There was a significant difference between the spleen indexes of the model control group, and the spleen indexes of high dose APS and the combination with high dose APS groups were significantly higher than that of the model control group (P<0.05).Compared with the DDP group, APS in various doses and combinations increased the thymus index and spleen index.Conclusions APS can improve the levels of cytokine IL -2, IL-6, IL-12 and TNF-αin mice bearing Lewis lung cancer, enhance the immune function impairment induced by DDP, has certain protective effect on the immune organs, and inhibit the growth of Lewis lung cancer in mice.When APS is used in combination with a half-dose of DDP, APS enhanced the inhibition of tumor growth.This mechanism may be related to the enhanced body immune function.Our results indicate that APS enhances the therapeutic effect of DDP and reduces its toxicity, therefore, may have potential application value in future treatment of solid tumors.

Objective To investigate the nature of the suprachoroidal fluid by detecting the concentration of total protein (TP), lactate dehydrogenase (LDH), albumin (ALB), total cholesterol (CHOL), total bilirubin (TBIL) in suprachoroidal liquid of patients who have rhegmatogenous retinal detachment with choroid detachment (RRDCD). Methods Eighteen RRDCD patients (18 eyes) who underwent vitrectomy were enrolled in this study. There were 10 males (10 eyes) and 8 females (8 eyes), 8 right eyes and 10 left eyes. There were 8 patients with age of ≤55 years, 10 patients with age of >55 years. There were 7 patients with duration of≤30 days, 11 patients with duration of >30 days. There were 7 eyes with diopters of ≥-6.0 D, 11 eyes with diopters of <-6.0 D. There were 11 eyes with class C proliferative vitreoretinopathy (PVR), 7 eyes with class D PVR. Suprachoroidal fluid samples were collected from all the patients, and took preoperative serum samples as RRDCD group. Ten serum samples of normal people were set as control group. The concentration of TP, LDH, ALB, CHOL, TBIL in all the subjects were measured. The properties of the suprachoroidal fluid were identified by Light standard and concentration standard of ALB, CHOL, TBIL. Results There was no difference on the concentration of TP, LDH, ALB, CHOL, TBIL from suprachoroidal fluid samples in the patients with different age, sex, eyes, diopter, PVR grade (P>0.05). There was no difference on the concentration of TP, LDH, ALB, CHOL, TBIL from preoperative serum samples in the patients between RRDCD group and control group (P>0.05). There was no difference on the concentration of ALB and CHOL from suprachoroidal fluid samples and preoperative serum samples in the RRDCD patients (P>0.05), but there were significant differences on the concentration of TP, LDH, TBIL (P<0.05). According to the Light standard, there were 17 cases of exudates and 1 case of transudate. According to the concentration standard of ALB, CHOL and TBIL, there were 14, 18, and 16 cases of exudates, and 4, 0, and 2 cases of transudate, respectively. There was no difference on the identification result of Light standard and concentration standard of ALB, CHOL, TBIL (χ2=2.090, 1.029, 0.364;P>0.05). Conclusion The suprachoroidal fluid of RRDCD patients composed of TP, LDH, CHOL and TBIL. The suprachoroidal fluid is more likely to be exudate.

Objective:To evaluate the role of hippocampal β-amyloid 42 (Aβ 42) deposition-induced accumulation of neutrophils in blood-brain barrier damage caused by sevoflurane anesthesia in aged rats. Methods:Seventy-two healthy male Wistar rats in which IT catheters were successfully planted, aged 18-20 months, weighing 600-650 g, were divided into 4 groups ( n=18 each) using a random number table method: control group (group C), γ-secretase inhibitor DAPT group (group D), sevoflurane anesthesia group (group S) and DAPT plus sevoflurane anesthesia group (group DS). Dimethyl sulfoxide 10 μl was intrathecally injected in group C and group S, and 30 min later group C inhaled 60% oxygen for 2 h, and group S inhaled 3.6% sevoflurane and 60% oxygen for 2 h and tibial fracture surgery was performed at the same time.DAPT 10 μl was intrathecally injected in group D and group DS, and 30 min later group D inhaled 60% oxygen for 2 h, and group DS inhaled 3.6% sevoflurane and 60% oxygen for 2 h and tibial fracture surgery was performed at the same time.The fear conditioning test was performed at 12 h after the end of treatment in each group, and the ratio of time spent freezing was calculated.The rats were sacrificed after the end of behavioral test, and hippocampal tissues were removed for determination of the expression of Aβ 42, occludin and matrix metalloproteinase-9 (MMP-9) (by Western blot), neutrophil count (by immuno-histochemistry), and content of Evans blue (EB) (by EB staining). Results:Compared with group C, the ratio of time spent freezing was significantly decreased, the expression of Aβ 42 and MMP-9 was up-regulated, the expression of occludin was down-regulated, the neutrophil count and content of EB were increased in group S and group DS ( P<0.05), and no significant change was found in the parameters mentioned above in group D ( P>0.05). Compared with group S, the ratio of time spent freezing was significantly increased, the expression of Aβ 42 and MMP-9 was down-regulated, the expression of occludin was up-regulated, the neutrophil count and content of EB were decreased in group DS ( P<0.05). Conclusion:The mechanism by which sevoflurane anesthesia leads to postoperative cognitive dysfunction is related to hippocampal Aβ 42 deposition-induced accumulation of neutrophils and causing damage to blood-brain barrier in aged rats.

Objective:To investigate the differential expression profile of circular RNA (circRNA) in high glucose-cultured human retinal vascular endothelial cells (HRVECs).Methods:HRVECs were divided into high glucose group, normal control group and hypertonic control group, and were cultured in 25 mmol/L glucose medium, 5.5 mmol/L glucose medium and 19.5 mmol/L mannitol+ 5.5 mmol/L glucose medium for 24 hours accordingly.The differentially expressed circRNA molecules between high glucose group and normal control group were screened by circRNA microarray analysis.The expression of the most significant differentially expressed circRNAs in different groups was verified by real-time quantitative PCR.The possible microRNA (miRNA) targets were analyzed through the Circular RNA Interactome database.Results:It was found that 448 circRNAs were differentially expressed (FC≥1.5 or FC≤0.67, P<0.05) in high glucose-cultured HRVECs, among which 182 were up-regulated and 266 were down-regulated.The top 3 significantly up-regulated circRNAs were hsa_circ_0002938, hsa_circ_0008036, and hsa_circ_0001946, and the top 3 significantly down-regulated circRNAs were hsa_circ_0035277, hsa_circ_0008344, and hsa_circ_0001874.Compared with normal control group and hypertonic control group, the relative expressions of top 3 up-regulated circRNAs were significantly enhanced and the relative expressions of top 3 down-regulated circRNAs were significantly reduced in high glucose group, showing statistically significant differences (all at P<0.05).No significant difference was found in the differentially expressed circRNAs between normal control group and hypertonic control group (all at P>0.05). Conclusions:CircRNAs are differentially expressed in high glucose-cultured HRVECs, and the differentially expressed circRNAs may be involved in the regulatory mechanism of diabetic retinopathy.

Objective To evaluate the role of autophagy in remifentanil-induced hyperalgesia in the rats with incisional pain.Methods Forty pathogen-free healthy male Sprague-Dawley rats,aged 2-3 months,weighing 250-280 g,were divided into 5 groups (n =8 each) using a random number table:control group (group C),remifentanil group (group R),incisional pain group (group Ⅰ),incisional pain plus remifentanil group (group IR) and incisional pain plus remifentanil plus autophagy inhibitor 3-methyladenine (3-MA) group (group MA).In group R,remifentanil was infused for 60 min at a rate of 1 μg · kg-1 · min-1 through the caudal vein.In group IR,a 1-cm longitudinal incision was made through skin,fascia and muscle of the plantar aspect of both hindpaws in anesthetized rats at 10 min of remifentanil infusion.In group MA,3-MA 15 mg/kg was injected intraperitoneally at 1 h before establishment of the model,and the other treatments were similar to those previously described in group IR,while the equal volume of normal saline was given in the other groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion and 2,6,24 and 48 h after infusion (T0-4).The rats were sacrificed after the last measurement of pain threshold at T4,and the L4-6 segments of the spinal cord were removed for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ),Beclin-1 and P62 by Western blot.Results Compared with group C,the MWT was significantly decreased and the TWL was shortened at T1-4 in R,Ⅰ,IR and MA groups,the expression of LC3 Ⅱ and Beclin-1 was up-regulated,and P62 expression was down-regulated in R and IR groups,the expression of LC3 Ⅱ was up-regulated at T4 in group Ⅰ,and the expression of LC3 Ⅱ and Beclin-1 was up-regulated at T4 in group MA (P＜0.0.5).Compared with group R,the MWT was significantly decreased and the TWL was shortened at T1-4,and the expression of LC3 Ⅱ was up-regulated at T4 in group IR (P＜0.05).Compared with group Ⅰ,the MWT was significantly decreased and the TWL was shortened at T1-4,and the expression of LC3 Ⅱ and Beclin-1 was up-regulated and P62 expression was down-regulated at T4 in group IR (P＜0.05).Compared with group IR,the MWT was significantly decreased and the TWL was shortened at T1-4,and the expression of LC3 Ⅱ and Beclin-1 was down-regulated and P62 expression was up-regulated at T4 in group MA (P＜0.05).Conclusion Autophagy is involved in the development and maintenance of remifentanil-induced hyperalgesia in the rats with incisional pain.

Objective:To evaluate the relationship between autophagy and oxidative stress during remifentanil-induced hyperalgesia in the rats with incisional pain.Methods:Thirty-two clean-grade healthy male Sprague-Dawley rats, weighing 230-250 g, aged 2-3 months, were divided into 4 groups ( n=8 each) using a random number table method: incisional pain group (I group), remifentanil + incisional pain group (RI group), autophagy inhibitor 3-methyladenine + remifentanil + incisional pain group (MRI group) and autophagy agonist rapamycin group + remifentanil + incisional pain group (RRI group). The model of incision pain was developed and the equal volume of normal saline was intravenously infused simultaneously for 60 min.In RI, MRI and RRI groups, the model of incision pain was developed and remifentanil 1 μg·kg -1·min -1 was simultaneously infused for 60 min.In MRI group, 3-methyladenine 15 mg/kg was intraperitoneally injected at 12 h before developing the model, and rapamycin 10 mg/kg was intraperitoneally injected at 12 h before developing the model in RRI group.Thermal paw withdrawal latency (TWL) and mechanical paw withdrawal threshold (MWT) were measured at 24 h before infusion of remifentanil or normal saline (T 0) and at 2, 6, 24 and 48 h after the end of infusion (T 1-4). The rats were sacrificed after the end of behavioral testing, and the lumbar enlargement segments of the spinal cord were harvested for determination of the expression of autophagy microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ), Beclin-1 and P62 (by Western blot), activity of superoxide dismutase (SOD) (by xanthine oxidase method) and content of malondialdehyde (MDA) (by thiobarbital method). Results:Compared with the baseline at T 0, PWT was significantly decreased and TWL was shortened at T 1-4 in the four groups ( P<0.05). Compared with I group, MWT was significantly decreased and TWL was shortened at T 1-4, the expression of LC3 Ⅱand Beclin-1 was up-regulated, the expression of P62 was down-regulated, SOD activity was decreased, and MDA content was increased in RI group ( P<0.05). Compared with RI group, MWT was significantly decreased and TWL was shortened at T 1-4, the expression of LC3 Ⅱand Beclin-1 was down-regulated, the expression of P62 was up-regulated, SOD activity was decreased, and MDA content was increased in MRI group ( P<0.05), and MWT was significantly increased and TWL was prolonged at T 1-4, the expression of LC3 Ⅱand Beclin-1 was up-regulated, the expression of P62 was down-regulated, SOD activity was increased, and MDA content was decreased in RRI group ( P<0.05). Conclusions:Autophagy is involved in the process of remifentanil-induced incisional hyperalgesia through regulating the level of oxidative stress in rats.