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Objective:To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells.Methods:SARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10 plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. Results:DNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed that S-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences ( t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1 % and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant ( t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant ( t=4.91, P=0.008). Conclusion:Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.
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Objective:To investigate the effectiveness and safety of the allogeneic corneal stromal flap in reducing the exposure of the drainage catheter in the implantation of Ahmed glaucoma valve.Methods:Fifty-three patients(61 eyes) with refractory glaucoma treated by Ahmed glaucoma valve implantation from January 2017 to April 2019 were retrospectively analysed. The patients were divided into sclera tunnel group(routine group) and sclera tunnel with allogeneic corneal stroma flap covering(improved group). The cumulative success rate of surgery, preoperative intraocular pressure(IOP), IOP at the last outpatient follow-up, the best-corrected visual acuity(BCVA), BCVA at the last outpatient follow-up, drainage catheter exposure and other surgical complications were collected from both groups at 6, 12 and 24 months after surgery. The data between the 2 groups were statistically analyzed, P<0.05 was statistically significant. Results:The cumulative success rates of 6, 12 and 24 months after operation were 89.7%, 86.2% and 69.0% in the routine group, and 90.6%, 90.6% and 71.8% in the improved group, respectively. There was no significant differences between the 2 groups( P>0.05). The IOP and BCVA at the final follow-up were significantly improved in both groups, with a statistical significant difference( P<0.05). In the conventional group, the exposure of drainage catheter occurred in 4 eyes(13.8%). Further surgeries were carried out to cover the exposed drainage catheters with allogeneic corneal stroma flaps and amniotic membrane and all had good recovery. There was no drainage catheter exposure in the improved group. The difference between the 2 groups was statistically significant( χ2=4.724, P=0.030). There was no significant difference in other surgical complications between the 2 groups( χ2=0.160, P=0.689). No intraoperative or postoperative complications regarding the corneal stromal flap were observed. Conclusion:Implantation of Ahmed glaucoma valve with allogeneic corneal stromal flap sealing can effectively reduce the exposure of the drainage catheter. It is a safe method.
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Objective:To explore the inhibitory effect of long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) by targeting microRNA-199a-5p (miR-199a-5p) on the apoptosis of human lens epithelial cells (LECs).Methods:The anterior lens capsule tissue of 23 age-related cataract patients who underwent cataract surgery in Xinxiang First People's Hospital from December 2018 to August 2019 was collected.At the same time, anterior lens capsules from 20 healthy donor were collected.The expressions of KCNQ1OT1 and miR-199a-5p in the tissues were detected by real-time fluorescence PCR.Human LECs SRA01/04 cultured in vitro were divided into blank control group, model control group, small interfering RNA-negative control (siR-NC) group, siR-KCNQ1OT1 group, miR-NC group, miR-199a-5p group, siR-KCNQ1OT1+ anti-miR-NC group and siR-KCNQ1OT1+ anti-miR-199a-5p group.No intervention was administered to blank control group.Cells in model control group were cultured with 100 μmol/L H 2O 2 for 24 hours to establish oxidative stress injured model, and cells in the other six groups were transfected with corresponding transfection reagents for 6 hours by liposome method according to grouping, and then treated with 100 μmol/L H 2O 2 for 24 hours.The expressions of KCNQ1OT1 and miR-199a-5p in lens anterior capsule tissue and LECs cells were determined by real-time fluorescent quantitative PCR.Cell viability was detected with thiazolyl blue (MTT). Cell apoptosis was analyzed by flow cytometry.The expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2 related X protein (Bax) proteins were assayed by Western blot.The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between KCNQ1OT1 and miR-199a-5p was verified by dual luciferase reporter experiment.The study protocol was approved by an Ethics Committee of Xinxiang First People's Hospital (No.2019-001). Written informed consent was obtained from relatives of patient. Results:The relative expression of KCNQ1OT1 in the anterior capsule of patients with age-related cataract was 2.41±0.42, which was significantly higher than 0.97±0.19 of normal people, and the relative expression of miR-199a-5p in the capsule of patients with age-related cataract was 0.36±0.12, which was lower than 1.04±0.15 of normal people, and the differences were statistically significant ( t=14.112, 16.507; both at P<0.001). Compared with blank control group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content were significantly increased, and the relative expressions of miR-199a-5p and bcl-2 protein, cell viability and SOD activity were significantly reduced in model control group, showing statistically significant differences (all at P<0.001). Compared with siR-NC group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content in cells of siR-KCNQ1OT1 group were decreased, while the relative expression of bcl-2 protein, cell survival rate and SOD activity were increased, and the differences were statistically significant (all at P<0.05). Compared with miR-NC group, the KCNQ1OT1-wild type (WT) luciferase activity in miR-199a-5p group was significantly decreased, with a statistically significant difference ( t=21.131, P<0.001). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly increased, and the relative expression of bax protein, cell apoptosis rate and MDA content were significantly decreased in miR-199a-5p group than those in miR-NC group, and the differences were statistically significant (all at P<0.05). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly lower, and the cell apoptosis rate, relative expression of bax protein and MDA content were significantly higher in siR-KCNQ1OT1+ anti-miR-199a-5p group than those in siR-KCNQ1OT1+ anti-miR-NC group, and the differences were statistically significant (all at P<0.05). Conclusions:The inhibition of KCNQ1OT1 can promote the cell viability of human LECs, inhibit H 2O 2-induced cell apoptosis and oxidative stress, and the mechanism may be related to the up-regulation of miR-199a-5p.
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Objective:To investigate the inhibitory effect of melatonin on pyroptosis of lens epithelium cells (HLECs) induced by hydrogen peroxide (H 2O 2) and its mechanism. Methods:The cultured HLECs were divided into normal control group, model control group, melatonin group, vitamin E group, and vitamin E solvent group.Cells in melatonin group, vitamin E group and vitamin E solvent group were pre-cultured with 1×10 -6 mol/L melatonin, 100 μmol/L vitamin E or equal volume of vitamin E solvent, then cultured with 100 μmol/L H 2O 2, respectively, and the cells in the normal control group and model control group were cultured with normal condition or 100 μmol/L H 2O 2, respectively.The HLECs transfected with nuclear factor erythroid 2-related factor 2 short hairpin RNA (shNrf2) or shNrf2 negtive control lentivirus and following with 100 μmol/L H 2O 2 treatment were served as shNrf2 group and shNrf2 negative control group, respectively; and the transfected cells treated with 1×10 -6 mol/L melatonin and subsequent 100 μmol/L H 2O 2 treatment were served as melatonin+ shNrf2 group and melatonin+ shNrf2 negative control group.The activity of lactic dehydrogenase (LDH) and the expression levels of interleukin (IL)-1β and IL-18 in the culture supernatant were detected by the enzyme linked immunosorbent assay (ELISA) kit.The concentration of reactive oxygen species (ROS) was assessed by flow cytometry.The expression level of Nrf2, NLRP3, apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1 p20 and GSDMD-N proteins were evaluated by Western blot. Results:Compared with model control group, the activity of LDH and the concentrations of IL-1β and IL-18 were significantly decreased in melatonin group and vitamin E group, showing statistically significant differences (all at P<0.05). The ROS fluorescence intensities were 13 040.67±1 550.66 and 12 593.67±1 677.06 in melatonin group and vitamin E group, respectively, which were significantly lower than 18 310.33±1 248.01 in model control group (both at P<0.05). The relative expression levels of Nrf2 protein were 4.24±0.44 and 3.73±0.38 in melatonin group and vitamin E group, respectively, which were significantly higher than 2.28±0.34 in model control group, and the relative expression levels of NLRP3, ASC, caspase-1 p20 and GSDMD-N in melatonin group and vitamin E group were significantly decreased than model control group (all at P<0.05). The relative expression level of Nrf2 protein in shNrf2 group and melatonin+ shNrf2 group was significantly reduced, and the expression levels of LDH, IL-1β, IL-18, ROS content, NLRP3, ASC, caspase-1 p20 and GSDMD-N were significantly increased in comparison with shNrf2 negative control group and melatonin+ shNrf2 negative control group, respectively (all at P<0.05). Conclusions:Melatonin can inhibit the release of NLRP3 inflammasome by activating Nrf2, and has an inhibitory effect on the pyroptosis of HLECs.
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Objective To investigate the effects of decorin (DCN) on apoptosis and oxidative stress in human lens epithelial cells (LECs) under high glucose condition.Methods HLE-B3 cells were cultured in vitro and the effect of DCN with different concentrations on HLE-B3 viability was determined by using cell counting kit-8 (CCK-8).The cultured cells were divided into normal control group,DCN group,high glucose group and DCN + high glucose group.Flow cytometry was used to detect the apoptosis rate and the expression of reactive oxygen species (ROS) in the cells.Microplate spectrophotometer was used to measure total superoxide dismutase (SOD) enzyme activity and the radio of glutathione (GSH)/glutathione disulfide (GSSG).Western blot was used to detect the expressions of bax and bcl-2 proteins.Results HLE-B3 cells were spindle shaped,with centered and clearly visible nuclei and neatly cell arrangment.According to CCK-8 method,survival rates of HLE-B3 in all groups were more than 90％.Different concentrations of DCN showed no significant effect on HLEoB3 survival rate (all at P＞0.05).After 48 hours of cell culture,the apoptosis rate of high glucose group was significantly higher than that of normal control group,and the apoptosis rate of DCN+high glucose group was significantly lower than that of high glucose group (both at P =0.000).The mean fluorescence intensity of intracellular ROS in the high-glucose group was significantly higher than that in the normal control group,and the mean fluorescence intensity of ROS in the DCN group was significantly higher than that in the high glucose group (both at P=0.000).The total SOD activity in the high glucose group was significantly lower than that in the normal control group and DCN group (P =0.007,0.004).The GSH/GSSG ratio of the high-glucose group was significantly lower than that of the normal control group and DCN group (both at P=0.000).Conclusions DCN can inhibit the apoptosis and oxidative stress of HLE-B3 under high glucose,which provides the basis for the treatment of diabetic cataract.
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Objective An analytical method for rapid screening and confirmation of multiple pesticide residues in vegetable and fruit was established according to GB 2763-2014 national food safety standard-maximum residue limits for pesticides in food,using ultra performance liquid chromatography-Q-time of flight mass spectrometry (UPLC-Q-TOF-MS).Methods Pesticide residues in vegetable and fruit were screened by comparing the accurate mass,isotope distribution and abundance in the accurate mass database,and confirmed by comparing with the spectra in the spectra library or by the spectral analysis method.Results The method was verified by spiked samples,and all pesticides were detected at the addition level of 10.0 and 50.0 μg/kg.The recovery of 90％ of the pesticides was in the range of 70％-120％,with relative standard deviation (RSD) ≤ 20％ (n =5).The method was applied to screen pesticides in 30 samples of vegetables and fruits.Twenty-seven pesticides were found and 2 samples exceeded the limit.Conclusion The method was sensitive,accurate and reproducible.Hundreds of pesticides in GB 2763-2014 could be screened and confirmed in a short period of time even without standard substance.It could provide an effective method for food safety control.
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Objective To explore the decision-making impulsivity in patients with idiopathic generalized epilepsy.Methods 39 patients with idiopathic generalized epilepsy and 40 healthy controls completed delay discounting task.The participants were demanded to make a series of choices between two different rewards after a delayed period (a smaller sooner reward or a larger longer reward).Results The delay discount rate k was transformed to common logarithm lg (k),and lg (k)=-1.75±0.86 in IGE group was more larger than that in HC group lg (k)=-2.21±0.72(t=2.58,P=0.01).IGE group performed worse than HC group in verbal fluency test-semantic (M (P25,P75):16.00 (14.00,19.00) vs 18.00 (16.00,22.75),Z =-2.86,P＜0.01),verbal fluency test-voice (M (P25,P75):4.00 (3.00,6.00) vs 7.00 (6.00,10.00),Z =-4.26,P＜0.01) and digital span backward test (M(P25,P75):5.00(5.00,7.00) vs 6.00 (5.00,8.00),Z=-2.48,P=0.01).In addition,lg (k) had significant correlation with verbal fluency test-semantic (r=0.32,P=0.048).Conclusion IGE group prefer immediate rewards and show more impulsive than HC group in delay discounting task.IGE group has cognitive deficit in frontal lobe language function and attention function.In addition,impulsivity is correlated with frontal lobe function.
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Objective To investigate the impairment of intertemporal choices in adolescents with positive and negative schizophrenic symptoms.Methods 30 adolescent schizophrenia patients with positive symptoms (positive symptoms group),30 adolescent schizophrenia patients with negative symptoms (negative symptoms group)and 30 healthy controls were selected.All the subjects were investigated with intertemporalchoice Test.Results (1) Under now conditions ((37.22±30.92)％,(19.67±16.35)％,P＜0.05)),notnow conditions ((35.74±31.69)％,(19.33± 18.07)％,P＜0.05)) and overall condition ((36.48±30.44) ％,(19.50± 13.82)％,P＜0.05)),the ratio of later-large (LL) choice in negative symptoms group were significantly higher than those in the healthy controls.Under now conditions ((37.22±30.92) ％,(20.37±22.33) ％,P＜0.05)),not-now conditions ((35.74± 31.69) ％,(22.04±22.05) ％,P＜ 0.05)) and overall condition ((36.48±30.44) ％,(21.20±21.57) ％,P＜0.05)),the ratio of LL choice in negative symptoms group were significantly higher than those in positive symptoms group.There were no differences in the ratio of LL choice between positive symptoms group and healthy controls (P＞ 0.05).(2)Pearson correlation analysis showed that the Vocabulary Fluency Test of negative symptoms group was positively correlated with LL selection ratio under now conditions (r=0.411,P=0.024).Conclusion The ability of intertemporal choices in adolescents schizophrenia patients with negative symptoms is impaired remarkably,while this kind of ability is impaired unremarkable in adolescence with negative symptoms.The ability of intertemporal choices in adolescents schizophrenic patients with negative symptoms is correlated with cognitive executive function.
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Objective To investigate the clinicopathological significance of transforming growth factor-β1 (TGF-β1), transforming growth factor-β1 receptor Ⅱ (TGF-βRⅡ ), Smad4 and Smad7 protein expressions in gastric carcinomas. Methods 109 cases of gastric carcinomas,28 of high grade dysplasia,20 of low grade dysplasia,30 of chronic atrophic gastritis,29 of intestinal metaplasia, and 21 of normal gastric mucosa were selected for the detection of the expression level of TGF-β1,TGF-βRⅡ , Smad4 and Smad7 proteins by immunohistochemistry. Results The expression of TGF-β1,TGF-βRⅡ and Smad7 proteins in gastric carcinomas and dysplasia (high and low grade) were significantly higher than those in chronic atrophic gastritis, intestinal metaplasia and normal gastric mucosa (all P <0. 001 ) ,whereas the expression of Smad4 was significantly increased in high and low grade dysplasia (cytoplas-mic IS :4.89±2.38,5.80±1.54, respectively; nuclear IS: 3.89± 1.52,3.80±1.33, respectively), and decreased in gastric carcinomas (cytoplasmic IS:2.41±2.27,nuclear IS:2.02±2. 14) (P <0.001 ,P <0.001 ,respective-ly). Meanwhile,the expressions of TGF-β1,TGF-βRⅡ and Smad7 were higher in advanced gastric cancer (4.36± 2.66,3.05±1.93,4.84±3.06, respectively) than those in early gastric cancer ( 2.93±1.85,2. 17±1.87, 4. 14±2.46, respectively) ( P < 0.01, P < 0.05, P < 0. 05, respectively ) ; however the expression of Smad4 protein was much higher in early gastric cancer (P < 0. 001 ). The expression of Smad4 protein was significantly correlated with tumor size( P < 0.05 ), lymph node metastasis ( P < 0. 001 ), T stage ( P < 0. 001 ) and chnical stage ( P < 0.001) ,and the expressions of TGF-βRⅡ and Smad4 proteins were correlated with patients' 5-year survival rate (P<0.05 ,P < 0.01 ). TGF-β1 showed the positive correlation with TGF-βRⅡ and Smad7 (r1= 0.45 ,P <0. 05; r2 = 0.49, P <0. 05), but negative correlation with Smad4 ( r = - 0.21,P < 0.05 ). Conclusion TGF-β1, TGF-βR Ⅱ, Smad4 and Smad7 protein may play an important role in the carcinogenesis of gastric carcinoma,and smad4 is the key in TGF-β pathway. Also, the detection of these proteins expression can be a useful marker for predicting the gas-tile cancer invasion,metastasis and patients' prognosis.