AIM: To investigate the effect of interleukin-8(IL-8)on the regulation of monocyte chemotactic protein-1(MCP-1)secreted by lens epithelial cells(LEC)during cell migration in the development of posterior capsule opacification(PCO).METHODS: A rat lens capsule model was established and cultured in medium supplemented with 10% fetal bovine serum. Upon migration of LEC to 30%-50% of the posterior capsule, serum was removed. The capsule was subsequently divided into two groups: a control group and an IL-8(15 ng/mL)treatment group. LEC migration was captured at multiple time points. The secretion and mRNA expression of MCP-1 were quantified using ELISA and RT-qPCR, respectively. Immunofluorescence was used to assess MCP-1 expression in the different experimental groups. SRA01/04 cells were divided into three groups: control, IL-8(15 ng/mL), and IL-8(15 ng/mL)+200 μmol/L Bindarit(BND)groups, with migration measured by the Transwell assay. Additionally, SRA01/04 cells were divided into negative control(NC), NC+15 ng/mL IL-8, and 15 ng/mL IL-8+p65 siRNA groups, and MCP-1 secretion and mRNA expression were further analyzed by ELISA and RT-qPCR.RESULTS:LEC migration in the rat lens capsule cultured in vitro showed that the cells migration of the 15 ng/mL IL-8 group significantly increased at 48, 72 and 96 h(all P<0.05). ELISA results revealed that MCP-1 levels in SRA01/04 cells from the 15 ng/mL IL-8-treated group were markedly higher than those in the control group at both 12 and 24 h(all P<0.05). RT-qPCR analysis also demonstrated a significant increase in MCP-1 mRNA expression in the 15 ng/mL IL-8 group at both time points(all P<0.05). Immunofluorescence staining indicated greater MCP-1 expression in capsular epithelial cells of the 15 ng/mL IL-8 group at 24 h(P=0.007). Transwell assays further confirmed increased cell migration in the 15 ng/mL IL-8 group compared to the control group(P=0.001), while the migration reduced in the 15 ng/mL IL-8+200 μmol/L BND group compared to the 15 ng/mL IL-8 group(P=0.003). Moreover, ELISA and RT-qPCR results demonstrated a significant increase in MCP-1 secretion and mRNA expression in the NC+15 ng/mL IL-8 group at both 12 and 24 h compared to the NC group(all P<0.01). In contrast, MCP-1 secretion and mRNA expression were reduced in the 15 ng/mL IL-8+p65 siRNA group compared to the NC+15 ng/mL IL-8 group at both time points(all P<0.01).CONCLUSION: IL-8 promotes the migration of residual epithelial cells and regulates the secretion and expression of MCP-1 in LEC. The mechanism underlying IL-8's effects appears to be mediated through the activation of the NF-κB/p65 signaling pathway.

Objective:To investigate the anti-aging and antioxidant effect of the heat shock transcription factor 1 (HSF1) on human retinal pigment epithelial cells.Methods:Two HSF1-deficient ARPE cells (ARPE/Hsf1 -/-) were constructed by using the clustered regularly interspaced short palindromic repeat and associated protein 9 (CRISPR/Cas9) gene editing system and named H8, H9 konckout cell strains.Experiments were operated on the 3 cell strains: wild-type, H8 and H9 cells.The content of reactive oxygen species in ARPE-19 cell was measured by DHE probe staining combined with flow cytometry technology, and the cell cycle was measured by flow cytometry technology.The cell viability at different time points was measured using cell counting kit-8 (CCK-8).Crystal violet staining assay was used to measure the relative ratio of cell survival.SA-β-gal staining assay was used to detect the ratio of ARPE-19 senescent cells.The expressions of HSP70, HSP27, clusterin (CLU), p53, p21 and interleukin (IL)-1β proteins were measured by Western blot technology.The expressions of p53, p21, IL-6, IL-8, IL-1β and monocyte chemoattractant protein 1 (MCP1) mRNA were measured by quantitative real-time PCR technology.Relative expression of heat shock response protein under different heat shock treatment conditions and HSP90 inhibitor IPI504, relative survival with different concentrations of H 2O 2, relative expression of p21 protein after treatment with or without ROS scavenger N-acetylcysteine (NAC) were compared in each cell strain. Results:Gene sequencing showed that H8 and H9 cell strains successfully carried mutated genes.Western blot experiment results showed that H8 and H9 cell strains did not express HSF1 protein, and HSF1 was successfully knocked out in ARPE-19 cells.Compared with wild-type cell, the expression levels of HSP70, HSP27 and CLU proteins in H8 and H9 cell strains significantly decreased, with statistically significant differences (all at P<0.05), and no significant difference was found in the relative HSP90 protein expression level ( F=0.29, P>0.05).Under different heat shock stimulation and IPI504 induction, the HSP70, HSP27, and CLU protein levels significantly increased in wild-type cells compared with before treatment, and the HSP70, HSP27, and CLU protein levels were significantly lower in H8 and H9 cell strains than in corresponding treated wild-type cells (all at P<0.05).Compared with wild-type cell strains, cell viability significantly decreased in H8 and H9 cell strains at 24, 48, 72, and 96 hours (all at P<0.05).Compared with wild-type cell strains, the percentage of cells in G1 phase was significantly higher and the mRNA and protein levels of the cell cycle inhibitors p53 and p21 significantly increased in H8 and H9 strains, showing statistically significant differences (all at P<0.05), and the ratio of positive cells for SA-β-gal staining significantly increased, showing statistically significant differences (all at P<0.001).The relative expression of aging-related inflammatory factors IL-6, IL-8, IL-1β, and MCP1 mRNA decreased, and the differences were statistically significant (all at P<0.001).In addition, compared with wild-type cell strains, the content of reactive oxygen species (ROS) was higher in H8 and H9 cell strains, and the differences were statistically significant (all at P<0.001).The expression of p21 protein in H8 and H9 cell strains wtih NAC treatment decreased significantly compared with non-NAC treatment cells (both at P<0.05).Compared with wild-type cell strains, H8 and H9 cell viability decreased at 200, 400, 600, and 800 μmol/L H 2O 2 treatment conditions, and the differences were statistically significant (all at P<0.05). Conclusions:Knockdown of HSF1 can downregulate the expression of heat shock proteins, activate the ROS/P53/P21 pathway, induce senescence in RPE cells, and increase the sensitivity of RPE to oxidative stress stimuli.HSF1 may have anti-senescence and anti-oxidant regulatory effects in RPE cells.

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.

OBJECTIVE:To investigate clinical efficacy and safety of oral loratadine combined with physiological seawater na-sal irrigation in the treatment of intermittent allergic rhinitis. METHODS:Totally 300 patients with intermittent allergic rhinitis were chosen from the Second Hospital of Hebei Medical University during Jan. 2013-Jun. 2015,and then divided into group A,B,C ac-cording to lottery method,with 100 cases in each group. Group A was given Loratadine tablets 10 mg,po,qd. Group B received nasal irrigation with physiological seawater nasal spray,every morning and evening. Group C was given oral loratadine combined with physiological seawater nasal irrigation. Treatment courses of 3 groups lasted for 28 d. Clinical efficiencies of 3 groups were compared as well as symptom and sign scores,respiration function indexes and inflammatory factor levels before and after treat-ment,and the clinical recurrences were followed up for 12 months. RESULTS:The total response rates of group A,B,C were 80.00%,78.00%,96.00%,respectively,and that of group C was significantly higher than that of group A and B,with statistical significance(P<0.05). Before treatment,there was no statistical significance in symptom and sign scores,respiration function in-dexes and inflammatory factor levels among 3 groups(P>0.05). After treatment,symptom and sign scores,the rates of PEF diur-nal variation,TNF-α,INF-γ and IL-4 in 3 groups were significantly lower than before treatment,and the levels of PEF and IL-12 were significantly higher than before treatment. Above indexes of group C were significantly better than those of group A and B, with statistical significance (P<0.05). Clinical recurrence rates of group A,B,C were 21.00%,23.00%,6.00%,and group C was significantly lower than the group A and B,with statistical significance (P<0.05). There was no statistical significance in above indexes between group A and group B (P>0.05). CONCLUSIONS:Oral loratadine combined with physiological seawater nasal irrigation in treatment of intermittent allergic rhinitis can efficiently relieve the nasal symptoms and signs,improve expiratoryfunction,reduce the inflammatory response levels and be help-ful to reduce the long-term recurrence risk.

ObjectiveTo investigate the clinical effect of Dahuang Zhechong capsules combined with entecavir in the treatment of chronic hepatitis B (CHB) patients with liver fibrosis. MethodsA total of 100 CHB patients with liver fibrosis who visited or were hospitalized in Shenzhen Hospital of Peking University from October 2014 to January 2016 were enrolled and randomly divided into Western medicine group and combined treatment group, with 50 patients in each group. The patients in the Western medicine group were given entecavir, and those in the combined treatment group were given Dahuang Zhechong capsules in addition to entecavir. The course of the treatment was 48 weeks. The changes in liver function, HBV DNA load, four serum parameters of liver fibrosis, liver stiffness, and aspartate aminotransferase-to-platelet ratio index (APRI)/FibroIndex were observed in both groups. The t-test was used for comparison of continuous data between groups, and the chi-square test was used for comparison of categorical data between groups. ResultsBoth groups showed significant reductions in serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and HBV DNA load after 48 weeks of treatment (all P＜0.05). After treatment, the four serum parameters of liver fibrosis all returned to normal after treatment, and the serum levels of hyaluronic acid, type Ⅲ precollagen, and type IV collagen showed significant differences between the two groups (all P＜005), and the portal vein diameter, thickness of the spleen, liver stiffness and APRI/FibroIndex also showed significant differences between the two groups (both P＜0.05). The combined treatment group had a significantly higher overall response rate than the Western medicine group (920% vs 720%, χ2=6.775, P=0009). ConclusionDahuang Zhechong capsules combined with entecavir have a better effect in the treatment of CHB patients with liver fibrosis compared with entecavir alone.

Objective:To express and purify the fusion protein of GST-FILIP-1L and prepare its polyclonal antibody. Methods:The constructed recombinant expression vectors pGEX-4T3-FILIP-1L were transformed into Escherichia Coli BL21. FILIP-1L fusion protein was induced by IPTG and purified by Glutathion Sepharse 4B . The rabbit was immunized by the purified fusion protein,and pro-duced serum with anti-FILIP-1L antibody. The titer of polyclonal antibody was detected by ELISA, the anti-FILIP-1L polyclonal antibody was purified by Active and its combining specificity with FILIP-1L protein was further identified by Western blot. Results:The GST-FILIP-1L fusion protein was highly expressed in E. coli, and its specific polyclonal antibody was obtained after the immunization. The polyclonal antibody purified by Active Ester Agrose was able to combine specially with FILIP-1L protein and transformed FILIP-1L protein in 293 cells and FILIP-1L protein of liver cancer cells, respectively. Conclusion: The GST-FILIP-1L fusion protein was expressed successfully,the anti-GST-FILIP-1L polyclonal antibodies with high titer and specificity are successfully prepared,these antibodies provide an useful experimental tool to research the biological function of the FILIP-1L protein.

Objective To assess the efficacy and safety of peginterferon ( PegIFN) α-2b in treatment of HBeAg-positive chronic hepatitis B ( CHB).Methods Thirty two patients with HBeAg-positive CHB admitted in Peking University Shenzhen Hospital during November 2013 and January 2014 were recruited in the study.Patients were center randomly assigned into two groups : 22 patients in test group were treated with 180 μg PegIFN α-2b, 1 /w for 48 wk; 10 patients in control group were treated with 180 μg PegIFN α-2a (Pegasys), 1 /w for 48wk.All patients were followed up for 24wk after treatment.Virology markers, HBV DNA levels and liver functions were monitored regularly , and adverse events were observed . Fisher’s exact test was used to compare the efficacy and safety between two groups .Results There were no statistically significant differences between the control group and test group in ALT normalization rates , HBV DNA negative rates and HBeAg serological conversion rates both at the end of treatment and at the end of 24-wk follow-up (all P >0.05).Both groups had similar adverse effect incidence rates (P >0.05), but retina disease occurred in 7 cases of test group, which was not observed in control group .Conclusion Compared with PegIFN α-2a, PegIFN α-2b has similar efficacy and safety for patients with HBeAg -positive CHB.

AIM:To clarify the impact of heart shock factor 4b (Hsf4b) K65R mutation on the regulation of downstream protein expression .METHODS:Non-functional Lys mutant plasmid pWZL-blast-HA-Hsf4b/K65R was genera-ted by replacing single , homologous amino acids using KOD-Plus-Mutagenesis-Kit.Mouse lens epithelial mLEC stable cell lines expressing Hsf4b or Hsf4b/K65R were constructed by lentivirus infection .The expression of Hsf4b in the mutant and the wildtype mLEC cells was confirmed by Western blotting .The expression of Hsf4b downstream proteins such as heat shock protein ( Hsp)70, Hsp90, Hsp27 and CryAB was examined by Western blotting and real-time PCR.RESULTS:The results of PCR and DNA sequencing confirmed the successful construction of mLEC Hsf 4b/K65R mutant.The K65R mutation didn’t influence Hsf4b expression in the mLEC cells.After K65R mutation in Hsf4b, the expression levels of Hsp27 and CryAB were down-regulated and the expression of Hsp 70i and Hsp90a upregulated.CONCLUSION: pWZL-blast-HA-Hsf4b/K65R can be used to construct a stable cell line by infecting with lentivirus .Hsf4b/K65R mutation influ-ences the regulation of downstream heat shock proteins .

Objective To observe the effects of magnesium isoglycyrrhizinate (MgIG) on the apoptosis of hepatocytes induced by simulated hypoxia reperfusion in rats and related mechanism.Methods Hepatocytes of the rats were isolated,cultured and identified,then,the liver cells in logarithmic growth phase were randomly divided into 4 groups:the hypoxia reperfusion injury model group,the low MgIG group (0.1 g/L),the moderate MgIG group (1 g/L) and the high MgIG group(10 g/L).Twenty-four hours after pre-treatment (exposure to simulated hypoxia for 6 h and re-perfusion for 4 h),following measurements were performed.① Changes in cell morphology were observed by optical microscopy.② Effects of MgIG on the vitality of hepatocytes were detected by MTT.③Apoptosis and rate of apoptosis were detected and calculated by AO/EB.④The expression levels of bcl-2 and Bax in all the groups were monitored by real time quantitative fluorescence PCR (RT-qPCR).Then,the detected data were analyzed by SPSS17.0.T test was applied for the comparison of the two mean sample lots,and single-factor ANOVA was used for the comparison of multiple samples.Results ①MTT assay indicated that MgIG at some concentrations (0.1,1 and 10 g/L)could improve the A value of the damaged hepatocytes induced by ischemia and re-perfusion(0.285 ± 0.017,0.320 ± 0.008 and 0.341 ± 0.019),as compared with that of the model group,with statistical significance (P ＜ 0.01).②AO/EB staining revealed that apoptosis rates in the low,moderate and high MgIG groups were(0.5763 ± 0.0405,0.2866 ±0.0619 and 0.1261 ±0.0439),which were all lower than that of the control group (0.7124 ± 0.0581),also with statistical significance (P ＜ 0.05).③RT-qPCR monitoring indicated that the expression levels of bcl-2 mRNA in the low,moderate and high MgIG groups (3.350 ± 0.151,9.279 ± 0.350 and 15.290±0.756) were all higher than that of the control group (0.928 ± 0.068),also with statistical significance(P ＜0.01).The expression levels of Bax mRNA in the low,moderate and high MgIG groups (0.743 ± 0.035,0.265 ± 0.065 and 0.119 ± 0.019)were all lower than that of the control group (0.945 ± 0.063),with statistical significance as well (P ＜ 0.01)Conclusions MgIG could increase the vitality of damaged hepatocytes induced by hypoxia-reperfusion and decrease rate of apoptosis,the mechanism of which might be associated with higher expression of bcl-2 mRNA and lower expression of Bax mRNA.

Objective To observe the effects of magnesium isoglycyrrhizinate (MgIG) on the apoptosis of hepatocytes induced by simulated hypoxia reperfusion in rats and related mechanism.Methods Hepatocytes of the rats were isolated,cultured and identified,then,the liver cells in logarithmic growth phase were randomly divided into 4 groups:the hypoxia reperfusion injury model group,the low MgIG group (0.1 g/L),the moderate MgIG group (1 g/L) and the high MgIG group(10 g/L).Twenty-four hours after pre-treatment (exposure to simulated hypoxia for 6 h and re-perfusion for 4 h),following measurements were performed.① Changes in cell morphology were observed by optical microscopy.② Effects of MgIG on the vitality of hepatocytes were detected by MTT.③Apoptosis and rate of apoptosis were detected and calculated by AO/EB.④The expression levels of bcl-2 and Bax in all the groups were monitored by real time quantitative fluorescence PCR (RT-qPCR).Then,the detected data were analyzed by SPSS17.0.T test was applied for the comparison of the two mean sample lots,and single-factor ANOVA was used for the comparison of multiple samples.Results ①MTT assay indicated that MgIG at some concentrations (0.1,1 and 10 g/L)could improve the A value of the damaged hepatocytes induced by ischemia and re-perfusion(0.285 ± 0.017,0.320 ± 0.008 and 0.341 ± 0.019),as compared with that of the model group,with statistical significance (P ＜ 0.01).②AO/EB staining revealed that apoptosis rates in the low,moderate and high MgIG groups were(0.5763 ± 0.0405,0.2866 ±0.0619 and 0.1261 ±0.0439),which were all lower than that of the control group (0.7124 ± 0.0581),also with statistical significance (P ＜ 0.05).③RT-qPCR monitoring indicated that the expression levels of bcl-2 mRNA in the low,moderate and high MgIG groups (3.350 ± 0.151,9.279 ± 0.350 and 15.290±0.756) were all higher than that of the control group (0.928 ± 0.068),also with statistical significance(P ＜0.01).The expression levels of Bax mRNA in the low,moderate and high MgIG groups (0.743 ± 0.035,0.265 ± 0.065 and 0.119 ± 0.019)were all lower than that of the control group (0.945 ± 0.063),with statistical significance as well (P ＜ 0.01)Conclusions MgIG could increase the vitality of damaged hepatocytes induced by hypoxia-reperfusion and decrease rate of apoptosis,the mechanism of which might be associated with higher expression of bcl-2 mRNA and lower expression of Bax mRNA.