AIM: To investigate the effects of insulin-like growth factor 1(IGF-1)on the secretion of transforming growth factor β2(TGF-β2), matrix metalloproteinase 2(MMP-2)and hypoxia-inducible factor 1α(HIF-1α)in human scleral fibroblasts(HSF)and their mechanism.METHODS: The cells were cultured with IGF-1 and PI3K/AKT pathway inhibitor LY294002, respectively. CCK-8 method was used to detect cell viability and determine the optimal concentration and time of drug action. Cell migration activity was observed by cell scratch method. To determine the effects of IGF-1 on HSF cells and the regulatory role of PI3K/AKT pathway, HSF cells were divided into control group(without drugs), IGF-1(80 μg/L)group, IGF-1+LY294002(80 μg/L+5 mmol/L)group, and LY294002(5 mmol/L)group, and were cultured for 24 h; the protein expression levels of TGF-β2, MMP-2, HIF-1α, PI3K and AKT were detected by Western blot; the fluorescence expression of TGF-β2, MMP-2 and HIF-1α was detected by cellular immunofluorescence.RESULTS: The results of CCK-8 showed that the cell viability of the 80 μg/L IGF-1 group cultured with different concentrations of IGF-1 was the highest(all P<0.05), and the cell viability of the 80 μg/L IGF-1 group at 24 h was the highest under different culture times. Therefore, the concentration of IGF-1 was selected as 80 μg/L for 24 h. The viability of cells cultured with different concentrations of LY294002 gradually decreased from 6 h(all P<0.05). According to the IC50 value, therefore, the concentration of LY294002 was selected as 5 mmol/L for 24 h. The cell scratch results showed that compared with the control group, the cell mobility of 40 μg/L and 80 μg/L IGF-1 groups was increased(all P<0.05). Compared with the control group, cell mobility in the 2.5 and 5 mmol/L LY294002 groups was decreased(all P<0.05). Western blot results showed that compared with the control group, the protein expressions of TGF-β2, MMP-2, HIF-1α, PI3K and AKT in the IGF-1 group were increased, while those in the LY294002 group were decreased(all P<0.05). Compared with the IGF-1 group, the expression levels of TGF-β2, MMP-2, HIF-1α, PI3K and AKT in the IGF-1+LY294002 group were decreased(all P<0.05). The results of cell immunofluorescence showed that compared with the control group, the fluorescence expressions of TGF-β2, MMP-2 and HIF-1α in the IGF-1 group were increased, while those in the LY294002 group were decreased(all P<0.05). Compared with the IGF-1 group, the fluorescence expressions of TGF-β2, MMP-2 and HIF-1α in the IGF-1+LY294002 group were significantly decreased(all P<0.05).CONCLUSION: IGF-1 promoted the proliferation and migration of human HSF. IGF-1 may up-regulate the expression of TGF-β2, MMP-2 and HIF-1α in HSF through the PI3K/AKT signaling pathway, and participate in the occurrence and development of myopia.

Objective To investigate the expression and role of T cell immunoglobulin and mucin domain-containing protein 3(Tim-3)in the pathogenesis of experimental autoimmune uveitis(EAU).Methods A total of 12 male C57BL/6J mice,aged 4 to 5 weeks,were selected and divided into the control group(n=3)and the experimental group(n=9)using a random number table.The control group(modeling time point:0 days after modeling)received no treatment,while the experimental group was induced to establish an EAU model(divided into three subgroups according to the modeling time points:7 days,14 days,and 21 days after modeling,with 3 mice in each subgroup).Firstly,the interphotoreceptor retinoid-binding protein 651-670 and complete Freund's adjuvant were fully mixed and emulsified.Then,the emulsion was subcutaneously injected into the two thighs,tail base,and neck of mice in the experimental group(each mouse received 200 μL of immune emulsion containing 500 pg of interphotoreceptor retinoid-binding protein 651-670).Subsequently,each mouse in the experimental group was also intraperitoneally injected with 1 μg of pertussis toxin.The anterior segment and fundus of mice in each group were observed and photographed under a slit-lamp microscope.The clinical and histopatho-logical scoring of these mice was conducted according to the Caspi grading scale based on the severity of inflammation.The serum levels of IFN-γ and IL-17 were measured using the enzyme-linked immunosorbent assay(ELISA),while the mRNA expression of Tim-3 in the spleen and ocular tissues was detected using the real-time quantitative polymerase chain reaction(RT-qPCR).Western blot was employed to detect the protein expression of Tim-3,and immunohistochemistry was used to examine the protein expression of Tim-3 in the spleen tissue.Statistical analysis was performed using GraphPad Prism 9.0.Results The clinical scores of the anterior segment,fundus,and histopathology of the mice increased over time after modeling,with statistically significant differences among these groups(P＜0.05).The serum levels of IFN-γ and IL-17 in the mice also increased over time after modeling,with statistically significant differences among these groups(P＜0.05).The relative mRNA expression of Tim-3 in the spleen and ocular tissues of the mice decreased over time after modeling,with statistically significant differences among these groups(P＜0.05).The protein expression of Tim-3 in the ocular and spleen tissues showed the same pattern as its mRNA expression.Conclusion The expression of Tim-3 decreases with the exacerbation of inflammation in the progression of EAU,suggesting that Tim-3 may play a negative immunoregulatory role in the development of uveitis.

Objective To investigate the expression and mechanistic role of the M2-type pyruvate kinase(PKM2)/signal transducer and activator of transcription 3(STAT3)signaling pathway in the development of experimental autoim-mune uveitis(EAU).Methods Eighteen 4-to 5-week-old C57BL/6J mice were selected and randomly divided into a control group(normal breeding,set as the 0-day state post-modeling),Experimental Group A,and Experimental Group B(both established as stable EAU mouse models using the interphotoreceptor retinoid-binding protein peptide 651-670,com-plete Freund's adjuvant,and pertussis toxin,designated as the 14-day and 21-day states post-modeling,respectively),with 6 mice in each group.The anterior segment of the mice was observed using a slit-lamp microscope,fundus findings were collected using a fundus camera,and clinical and histopathological scores were evaluated after hematoxylin-eosin stai-ning.The protein expression level of serum interleukin(IL)-17A was detected by ELISA.The expression level of PKM2 protein in retinal tissue was detected by immunohistochemistry.The protein expression levels of PKM2,phosphorylated STAT3(p-STAT3),and STAT3 in mouse retinal tissue were detected by Western blot.Results The clinical scores of the anterior segment and fundus,as well as the retinal histopathological scores in Experimental Group A and Experimental Group B,were all significantly higher than those in the control group(all P＜0.05).The serum IL-17A protein expression levels in the control group,Experimental Group A,and Experimental Group B were(69.05±0.45)ng·L-1,(75.06±0.46)ng·L-1,and(72.04±0.82)ng·L-1,respectively.The optical density values of PKM2 protein expression in retinal tissue were(18.51±2.59)％,(37.35±4.67)％,and(29.75±2.17)％,respectively.The expression levels of serum IL-17A protein,retinal PKM2 protein,and retinal STAT3 and p-STAT3 proteins in Experimental Group A and Experimental Group B were all significantly higher than those in the control group(all P＜0.05).Conclusion The expression levels of key factors in the PKM2/STAT3 signaling pathway are positively correlated with the severity of EAU,indicating that this sig-naling pathway,as a positive regulator of the immune response,is involved in the pathological process of EAU.

Objective:To study the risk factors and the molecular epidemiology characteristics for Carbapenem-resistant Enterobacteriaceae(CRE) colonization in neonatal inpatients in Shenzhen region, China, which provide reference for the prevention and control of clinical CRE infection.Methods:This study is a prospective case-control study.Anal samples from inpatients between January 2023 and December 2023 at Longgang Maternity and Child Institute of Shantou University Medical College and Shenzhen Children's Hospital were collected for screening CRE strain. Drug susceptibility test, modified Carbapenem Inactivation Method (mCIM) test, drug resistance-related gene sequencing and multilocus sequence typing (MLST) were performed for isolated CRE strains.Meanwhile, the clinical data were collected for analyzing the risk factors of CRE intestinal colonization by multivariate regression analysis.Results:A total of 1 517 patients were screened, 26 CRE(1.7%, 26/1 517) were identified which including 14 Escherichia coli(53.8%, 14/26), 11 Klebsiella pneumoniae(42.3%, 11/26), 1 Enterobacter cloacae(3.9%, 1/26). The predominant carbapenemase gene was New Delhi Metallo(NDM) (92.4%, 24/26), followed by Imipenem (IMP) (3.8%, 1/26) and Guiana extended spectrum gene (GES) (3.8%, 1/26).Among the carried NDM resistance genes, New Delhi Metallo 5 (NDM5) was the main one, accounting for 84.6% (22/26).The MLST typing of Escherichia coli was mainly Sequence Type 48 (ST48) (6/14), while that of Klebsiella pneumoniae was mainly Sequence Type 35 (ST35) (10/11). All CRE isolates were resistant to penicillin, penicillinase inhibitors, cephalosporins, ertapenem and imipenem.The resistance rates of Escherichia coli to amikacin, levofloxacin was 1/14, 4/14, respectively. All isolates of Klebsiella pneumoniae were sensitive to amikacin, and the resistance rate to levofloxacin is 1/11. Risk factors for CRE colonization include the older age, length of hospital stay, tracheal intubation, invasive respiration, lumbar puncture, Apgar <7 score, and exposure to antibiotics.Conclusions:NDM5 is the predominant resistant gene in CRE isolated from neonatal patients feces in Shenzhen region.It is necessary to strengthen the screening of CRE colonization in neonate for prevention and control of CRE infection.

Objective:To study the risk factors and the molecular epidemiology characteristics for Carbapenem-resistant Enterobacteriaceae(CRE) colonization in neonatal inpatients in Shenzhen region, China, which provide reference for the prevention and control of clinical CRE infection.Methods:This study is a prospective case-control study.Anal samples from inpatients between January 2023 and December 2023 at Longgang Maternity and Child Institute of Shantou University Medical College and Shenzhen Children's Hospital were collected for screening CRE strain. Drug susceptibility test, modified Carbapenem Inactivation Method (mCIM) test, drug resistance-related gene sequencing and multilocus sequence typing (MLST) were performed for isolated CRE strains.Meanwhile, the clinical data were collected for analyzing the risk factors of CRE intestinal colonization by multivariate regression analysis.Results:A total of 1 517 patients were screened, 26 CRE(1.7%, 26/1 517) were identified which including 14 Escherichia coli(53.8%, 14/26), 11 Klebsiella pneumoniae(42.3%, 11/26), 1 Enterobacter cloacae(3.9%, 1/26). The predominant carbapenemase gene was New Delhi Metallo(NDM) (92.4%, 24/26), followed by Imipenem (IMP) (3.8%, 1/26) and Guiana extended spectrum gene (GES) (3.8%, 1/26).Among the carried NDM resistance genes, New Delhi Metallo 5 (NDM5) was the main one, accounting for 84.6% (22/26).The MLST typing of Escherichia coli was mainly Sequence Type 48 (ST48) (6/14), while that of Klebsiella pneumoniae was mainly Sequence Type 35 (ST35) (10/11). All CRE isolates were resistant to penicillin, penicillinase inhibitors, cephalosporins, ertapenem and imipenem.The resistance rates of Escherichia coli to amikacin, levofloxacin was 1/14, 4/14, respectively. All isolates of Klebsiella pneumoniae were sensitive to amikacin, and the resistance rate to levofloxacin is 1/11. Risk factors for CRE colonization include the older age, length of hospital stay, tracheal intubation, invasive respiration, lumbar puncture, Apgar <7 score, and exposure to antibiotics.Conclusions:NDM5 is the predominant resistant gene in CRE isolated from neonatal patients feces in Shenzhen region.It is necessary to strengthen the screening of CRE colonization in neonate for prevention and control of CRE infection.

Objective To investigate the expression and role of T cell immunoglobulin and mucin domain-containing protein 3(Tim-3)in the pathogenesis of experimental autoimmune uveitis(EAU).Methods A total of 12 male C57BL/6J mice,aged 4 to 5 weeks,were selected and divided into the control group(n=3)and the experimental group(n=9)using a random number table.The control group(modeling time point:0 days after modeling)received no treatment,while the experimental group was induced to establish an EAU model(divided into three subgroups according to the modeling time points:7 days,14 days,and 21 days after modeling,with 3 mice in each subgroup).Firstly,the interphotoreceptor retinoid-binding protein 651-670 and complete Freund's adjuvant were fully mixed and emulsified.Then,the emulsion was subcutaneously injected into the two thighs,tail base,and neck of mice in the experimental group(each mouse received 200 μL of immune emulsion containing 500 pg of interphotoreceptor retinoid-binding protein 651-670).Subsequently,each mouse in the experimental group was also intraperitoneally injected with 1 μg of pertussis toxin.The anterior segment and fundus of mice in each group were observed and photographed under a slit-lamp microscope.The clinical and histopatho-logical scoring of these mice was conducted according to the Caspi grading scale based on the severity of inflammation.The serum levels of IFN-γ and IL-17 were measured using the enzyme-linked immunosorbent assay(ELISA),while the mRNA expression of Tim-3 in the spleen and ocular tissues was detected using the real-time quantitative polymerase chain reaction(RT-qPCR).Western blot was employed to detect the protein expression of Tim-3,and immunohistochemistry was used to examine the protein expression of Tim-3 in the spleen tissue.Statistical analysis was performed using GraphPad Prism 9.0.Results The clinical scores of the anterior segment,fundus,and histopathology of the mice increased over time after modeling,with statistically significant differences among these groups(P＜0.05).The serum levels of IFN-γ and IL-17 in the mice also increased over time after modeling,with statistically significant differences among these groups(P＜0.05).The relative mRNA expression of Tim-3 in the spleen and ocular tissues of the mice decreased over time after modeling,with statistically significant differences among these groups(P＜0.05).The protein expression of Tim-3 in the ocular and spleen tissues showed the same pattern as its mRNA expression.Conclusion The expression of Tim-3 decreases with the exacerbation of inflammation in the progression of EAU,suggesting that Tim-3 may play a negative immunoregulatory role in the development of uveitis.

Objective To investigate the expression and mechanistic role of the M2-type pyruvate kinase(PKM2)/signal transducer and activator of transcription 3(STAT3)signaling pathway in the development of experimental autoim-mune uveitis(EAU).Methods Eighteen 4-to 5-week-old C57BL/6J mice were selected and randomly divided into a control group(normal breeding,set as the 0-day state post-modeling),Experimental Group A,and Experimental Group B(both established as stable EAU mouse models using the interphotoreceptor retinoid-binding protein peptide 651-670,com-plete Freund's adjuvant,and pertussis toxin,designated as the 14-day and 21-day states post-modeling,respectively),with 6 mice in each group.The anterior segment of the mice was observed using a slit-lamp microscope,fundus findings were collected using a fundus camera,and clinical and histopathological scores were evaluated after hematoxylin-eosin stai-ning.The protein expression level of serum interleukin(IL)-17A was detected by ELISA.The expression level of PKM2 protein in retinal tissue was detected by immunohistochemistry.The protein expression levels of PKM2,phosphorylated STAT3(p-STAT3),and STAT3 in mouse retinal tissue were detected by Western blot.Results The clinical scores of the anterior segment and fundus,as well as the retinal histopathological scores in Experimental Group A and Experimental Group B,were all significantly higher than those in the control group(all P＜0.05).The serum IL-17A protein expression levels in the control group,Experimental Group A,and Experimental Group B were(69.05±0.45)ng·L-1,(75.06±0.46)ng·L-1,and(72.04±0.82)ng·L-1,respectively.The optical density values of PKM2 protein expression in retinal tissue were(18.51±2.59)％,(37.35±4.67)％,and(29.75±2.17)％,respectively.The expression levels of serum IL-17A protein,retinal PKM2 protein,and retinal STAT3 and p-STAT3 proteins in Experimental Group A and Experimental Group B were all significantly higher than those in the control group(all P＜0.05).Conclusion The expression levels of key factors in the PKM2/STAT3 signaling pathway are positively correlated with the severity of EAU,indicating that this sig-naling pathway,as a positive regulator of the immune response,is involved in the pathological process of EAU.

Objective To investigate the effects of insulin-like growth factor 1(IGF-1)on the expression of transfor-ming growth factor β2(TGF-β2)and matrix metalloproteinase 2(MMP-2)in human retinal pigment epithelial cells(ARPE-19)and related mechanisms.Methods ARPE-19 cells were cultured for 6 h,12 h,24 h and 48 h,respectively,with dif-ferent concentrations of IGF-1 and LY294002.The cell viability was detected using the cell counting kit-8 to determine the optimal action concentration and time of IGF-1 and LY294002.The cell migration activity was detected using the cell scratch assay.The concentration of TGF-β2 in cell culture supernatant was detected using the enzyme-linked immunosorbent assay(ELISA).ARPE-19 cells were divided into the control group,IGF-1 group(80 μg·L-1 IGF-1),IGF-1+LY294002 group(80 μg·L-1 IGF-1+30 mmol·L-1 LY294002),and LY294002 group(30 mmol·L-1 LY294002)and cultured with serum-free DMEM/F12 medium,while cells in the control group received no treatment.The mRNA and protein expression levels of TGF-β2,MMP-2,phosphoinositide 3-kinase(PI3K)and protein kinase B(AKT)in the cells were measured using the re-verse transcription-polymerase chain reaction(RT-PCR)and Western blot,respectively.Results Compared with the 0μg·L-1 IGF-1 group,the cell viability in the 80 μg·L-1 IGF-1 group changed the most significantly at 24 h(P＜0.05);thus,the optimal concentration of IGF-1 was 80 μg·L-1 and the optimal culture time was 24 h.Compared with the 0 mmol·L-1 LY294002 group,the inhibition concentration in the 30 mmol·L-1 LY294002 group at 24 h was close to half;thus,the optimal concentration of LY294002 was 30 mmol·L-1 and the optimal culture time was 24 h.The cell scratch assay re-sults showed that the cell migration rate was significantly different among the 0 μg·L-1 IGF-1 group,40 μg·L-1 IGF-1 group,and 80 μg·L-1 IGF-1 group(all P＜0.05).ELISA results showed that there was a statistically significant difference in the concentration of TGF-β2 in cell supernatant among the 0 μg·L-1 IGF-1 group,40 μg·L-1 IGF-1 group,and 80 μg·L-1 IGF-1 group(all P＜0.05).RT-PCR and Western blot results showed that after 24 h culture with IGF-1 and LY294002,compared with the control group,the mRNA and protein expression levels of TGF-β2,MMP-2,PI3K and AKT in the IGF-1 group increased,while the mRNA and protein expression levels of TGF-β2,MMP-2,PI3K and AKT in the LY294002 group decreased(all P＜0.05).Compared with the IGF-1 group,the mRNA and protein expression levels of TGF-[32,MMP-2,PI3K and AKT in the IGF-1+LY294002 group decreased(all P＜0.05).Conclusion IGF-1 can promote the proliferation and migration of ARPE-19 cells.IGF-1 may up-regulate the expression of TGF-β2 and MMP-2 in ARPE-19 cells through the PI3K/AKT signaling pathway and participate in the occurrence and development of myopia.

Objective To explore the effects of metformin on the proliferation,migration,and epithelial-mesenchy-mal transition(EMT)of human lens epithelial cells(LEC)induced by transforming growth factor-β2(TGF-β2).Methods Immortalized human LEC(HLEB-3 cells)was selected as the cell source.Human LEC with a cell fusion degree of 80％was cultured in DMEM low-glucose medium containing 10 mg·L-1 TGF-β2 for 24 hours as the control group.The cells treated with TGF-β2 and then further treated with different concentrations of metformin were used as the experimental group.After treatment,the morphological changes of cells in each group were observed under an inverted microscope.The cytotoxicity was detected by CCK-8 assay,and the cell survival rate was calculated.The expression levels of Yes-associated protein 1(YAP1),large tumor suppressor 1(LATS1),and Vimentin in cells were detected by Western blot.The mRNA ex-pression levels of YAP1,LATS1,mammalian STE20-like kinase 1(MST1),Vimentin,and E-cadherin were detected by re-al-time quantitative polymerase chain reaction.Results The cytotoxicity test of metformin showed that when the concen-tration of metformin was greater than 15 mmol·L-1,the survival rate of human LEC significantly decreased,indicating that the concentration of metformin had a significant impact on the survival of LEC.Therefore,15 mmol·L-1 was selected for subsequent experiments.Metformin significantly inhibited the proliferation of human LEC induced by TGF-β2 in a dose-dependent manner(P＜0.001).After 24 hours of treatment with 15 mmol·L-1 metformin,the relative expression levels of YAP1 and Vimentin proteins in human LEC were lower than those in the control group(both P＜0.05),while the relative expression level of LATS1 protein was higher than that in the control group(P＜0.05).After 24 hours of treatment with 15 mmol·L-1 metformin,the relative expression levels of YAP1 and Vimentin mRNA in human LEC were lower than those in the control group,while the relative expression levels of LATS1,MST1,and E-cadherin mRNA were higher than those in the control group,with statistically significant differences(all P＜0.05).Conclusion Metformin can inhibit the prolifer-ation,migration and EMT of human LEC induced by TGF-β2 in vitro,downregulate the expression of YAP1 and Vimentin mRNA,and upregulate the expression of LATS1,MST 1 and E-cadherin.The mechanism of action may be related to its ac-tivation of the Hippo signaling pathway.

Objective:To explore the regulation of eight weeks aerobic exercise on brain spontaneous activity in individuals with subthreshold depression.Methods:Through subject recruitment, a total of 44 subjects with subthreshold depression (subthreshold depression group, StD) who scored≥5 according to Patient Health Questionnare-9 (PHQ-9) but did not meet the diagnostic criteria for clinical depression, and 34 healthy subjects (health control group, HC) were included. Two groups of participants were given 8-week moderate-intensity aerobic exercise intervention, and resting state functional magnetic resonance imaging (rs-fMRI) data were collected before and after the intervention to analyze the fractional amplitude of low frequency fluctuations (fALFF) in the two groups. Horizontal two-sample t-tests with years of education as a covariate and longitudinal paired-samples t-tests were performed for fALFF in both groups at baseline and after 8-week moderate-intensity aerobic exercise intervention. The relationship between the fALFF values in significant difference brain regions and psycho-psychological scales were examined by Spearman correlation. Results:After 8 weeks of moderate intensity aerobic exercise, the scores of PHQ-9 and Self-Rating Anxiety Scale (SAS) in StD were significantly lower than baseline ( t=7.08,5.99, both P<0.001). There was no significant difference in fALFF value between StD and HC at baseline. After aerobic exercise intervention, compared with the HC, the fALFF value of the left parahippocampal gyrus in the StD was significantly decreased, and the fALFF value of the left superior frontal gyrus and left precentral gyrus was significantly increased. After aerobic exercise intervention, compared with the baseline, the StD had significantly higher fALFF values in the left inferior parietal lobe (supramarginal gyrus and angular gyrus), right supramarginal gyrus and left anterior cingulate gyrus. In addition, compared with the baseline, the fALFF value of the right precentral gyrus in the HC was significantly decreased. After aerobic exercise intervention, the fALFF value of the left inferior parietal lobe was negatively correlated with the SAS scores ( r= -0.31, P=0.038), the fALFF value of the left anterior cingulate gyrus and the PHQ-9 item 2 (low mood) scores was negatively correlated ( r= -0.41, P=0.006), and the fALFF values of the left inferior parietal lobe, right supramarginal gyrus and the PHQ-9 item 6 (low self-evaluation) scores were negative correlated ( r=-0.39, P=0.010; r=-0.30, P=0.048). Conclusion:Eight weeks of moderate-intensity aerobic exercise can regulate the spontaneous activity pattern of local brain regions in individuals with subthreshold depression, and fALFF analysis can provide objective evidence for the antidepressant effect of aerobic exercise.