Objective To investigate the effect of a G-protein receptor 110(GPR110)ligand on retinal neuroinflam-mation in diabetic mice by regulating the Janus kinase 2/signal transduction and activator of transcription 3(JAK2/STAT3)signaling pathway.Methods This experiment was divided into two parts.(1)Twelve C57BL/6J mice(24 eyes)were randomly divided into the control group,4-week streptozotocin(STZ)induction(STZ4-week)group,STZ 8-week group,and STZ 12-week group,with 3 mice in each group.The diabetic model was induced by intraperitoneal injection of STZ(150 mg·kg-1)in all groups except the control group.The expression of GPR110 and glutamine synthetase(GS)in the retinal tissue of mice was detected by immunofluorescence staining to screen the optimal acting time.(2)Thirty-two C57BL/6J mice(64 eyes)were divided into the control group,STZ 8-week group,the STZ+N-docosahexaenol ethanola-mine(SYN)group,and the STZ+SYN+adeno-associated virus-mediated GPR110(AAV-GPR110)group,with 8 mice in each group,and they were subjected to corresponding treatment.Immunohistochemistry was performed to detect the ex-pression of glial fibrillary acidic protein(GFAP)in mouse retinal tissues.ELISA was performed to detect the level of inter-leukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in mouse retinal tissues.Western blot was performed to detect the ratio of JAK2 to phosphorylated JAK2(p-JAK2)and that of STAT3 to phosphorylated STAT3(p-STAT3).Results(1)GPR110 in the retina of mice in the STZ 4-week group,STZ 8-week group,and STZ 12-week group was mainly expressed in Müller cells.The highest expression intensity of GPR110 was observed in the retina of mice in the STZ 8-week group;therefore,the diabetic mice in the STZ 8-week group were selected for subsequent experiments.(2)Compared with the control group,mice in the STZ 8-week group and the STZ+SYN+AAV-GPR110 group exhibited a decrease in the retinal thickness and the number of retinal ganglion cells,those in the STZ+SYN group displayed a de-crease in the retinal thickness(all P＜0.05),and those in the other three groups presented a significant increase in the in-tensity of GFAP-positive expression and an increase in the level of IL-6,IL-1β,and TNF-α and the p-JAK2/JAK2 and p-STAT3/STAT3 values(all P＜0.05).Compared with the STZ 8-week group,the retinal thickness and the number of retinal ganglion cells in the STZ+SYN group increased,the intensity of GFAP-positive expression decreased,and the level of IL-6,IL-1 β,and TNF-α and the p-JAK2/JAK2 and p-STAT3/STAT3 values decreased(all P＜0.05).Compared with the STZ+SYN group,mice in the STZ+SYN+AAV-GPR110 group showed a decrease in the retinal thickness and the number of retinal ganglion cells,a significant increase in the intensity of GFAP-positive expression,and an increase in the level of IL-6,IL-1 β,and TNF-α and the p-JAK2/JAK2 and p-STAT3/STAT3 values(all P＜0.05).Conclusion The GPR110 ligand could alleviate retinal neuroinflammation in diabetic mice by inhibiting the JAK2/STAT3 signaling pathway.

Objective To investigate the mechanism of serine protease inhibitor A3N(SerpinA3N)in alleviating reti-nal neural injury in diabetic mice by inhibiting Müller cell inflammation.Methods Thirty-six db/db mice(72 eyes)were randomly divided into the db/db group,the db/db+SerpinA3N-overexpressing adeno-associated virus(AAV-SerpinA3N)group,and the db/db+empty vector adeno-associated virus(NC-SerpinA3N)group,with 12 mice in each group.Twelve age-matched healthy male littermate mice were randomly selected as the healthy control group(db/m group).Mice in each group were sacrificed 4 weeks after the corresponding treatments.Immunofluorescence staining was used to detect the co-localization of SerpinA3N and glial fibrillary acidic protein(GFAP)in the mouse retina.Hematoxylin-eosin(HE)staining was used to observe histopathological changes in the retinal tissue.TUNEL staining was used to detect the apoptosis of reti-nal ganglion cells(RGCs).Immunohistochemistry was used to detect GFAP expression.ELISA was used to measure the levels of inflammatory factors[interleukin(IL)-1 β,IL-6,IL-18,and tumor necrosis factor-α(TNF-α)]in the retinal tis-sue.The predicted target genes of SerpinA3N were imported into the STRING database to construct a protein-protein inter-action(PPI)network.The highest-scoring target was selected based on the scores for molecular docking.Western blot was used to detect the expression levels of SerpinA3N,nuclear factor kappa B(NF-κB),and spleen focus-forming virus proviral integration oncogene(Spi1)proteins in the retinal tissue.Results Immunofluorescence staining showed co-lo-calized expression of SerpinA3N and GFAP in the retinal tissue.Compared with the db/m group,the db/db,db/db+NC-SerpinA3N,and db/db+AAV-SerpinA3N groups showed decreased retinal thickness and RGC count,and increased number of TUNEL-positive cells,relative GFAP-positive expression intensity,levels of all inflammatory factors,and expression lev-els of NF-κB and Spi1 proteins,while SerpinA3N protein expression was decreased(all P＜0.05).Compared with the db/db group,the db/db+AAV-SerpinA3N group showed increased retinal thickness and RGC count,and decreased number of TUNEL-positive cells,relative GFAP-positive expression intensity,levels of all inflammatory factors,and expression levels of NF-κB and Spi1 proteins,while SerpinA3N protein expression was increased(all P＜0.05).Compared with the db/db+AAV-SerpinA3N group,the db/db+NC-SerpinA3N group showed decreased retinal thickness and RGC count,and increased number of TUNEL-positive cells,relative GFAP-positive expression intensity,levels of all inflammatory factors,and ex-pression levels of NF-κB and Spi1 proteins,while SerpinA3N protein expression was decreased(all P＜0.05).The PPI re-sults indicated an interaction between SerpinA3N and Spi1.Molecular docking results showed that Spi1 could form hydro-gen bonds with residues surrounding SerpinA3N.Conclusion Overexpression of SerpinA3N can inhibit Müller cell in-flammation and ameliorate retinal neural injury in diabetic mice,and the mechanism may be associated with the inhibition of the Spi1/NF-κB signaling pathway.

Objective To investigate the effect of a G-protein receptor 110(GPR110)ligand on retinal neuroinflam-mation in diabetic mice by regulating the Janus kinase 2/signal transduction and activator of transcription 3(JAK2/STAT3)signaling pathway.Methods This experiment was divided into two parts.(1)Twelve C57BL/6J mice(24 eyes)were randomly divided into the control group,4-week streptozotocin(STZ)induction(STZ4-week)group,STZ 8-week group,and STZ 12-week group,with 3 mice in each group.The diabetic model was induced by intraperitoneal injection of STZ(150 mg·kg-1)in all groups except the control group.The expression of GPR110 and glutamine synthetase(GS)in the retinal tissue of mice was detected by immunofluorescence staining to screen the optimal acting time.(2)Thirty-two C57BL/6J mice(64 eyes)were divided into the control group,STZ 8-week group,the STZ+N-docosahexaenol ethanola-mine(SYN)group,and the STZ+SYN+adeno-associated virus-mediated GPR110(AAV-GPR110)group,with 8 mice in each group,and they were subjected to corresponding treatment.Immunohistochemistry was performed to detect the ex-pression of glial fibrillary acidic protein(GFAP)in mouse retinal tissues.ELISA was performed to detect the level of inter-leukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in mouse retinal tissues.Western blot was performed to detect the ratio of JAK2 to phosphorylated JAK2(p-JAK2)and that of STAT3 to phosphorylated STAT3(p-STAT3).Results(1)GPR110 in the retina of mice in the STZ 4-week group,STZ 8-week group,and STZ 12-week group was mainly expressed in Müller cells.The highest expression intensity of GPR110 was observed in the retina of mice in the STZ 8-week group;therefore,the diabetic mice in the STZ 8-week group were selected for subsequent experiments.(2)Compared with the control group,mice in the STZ 8-week group and the STZ+SYN+AAV-GPR110 group exhibited a decrease in the retinal thickness and the number of retinal ganglion cells,those in the STZ+SYN group displayed a de-crease in the retinal thickness(all P＜0.05),and those in the other three groups presented a significant increase in the in-tensity of GFAP-positive expression and an increase in the level of IL-6,IL-1β,and TNF-α and the p-JAK2/JAK2 and p-STAT3/STAT3 values(all P＜0.05).Compared with the STZ 8-week group,the retinal thickness and the number of retinal ganglion cells in the STZ+SYN group increased,the intensity of GFAP-positive expression decreased,and the level of IL-6,IL-1 β,and TNF-α and the p-JAK2/JAK2 and p-STAT3/STAT3 values decreased(all P＜0.05).Compared with the STZ+SYN group,mice in the STZ+SYN+AAV-GPR110 group showed a decrease in the retinal thickness and the number of retinal ganglion cells,a significant increase in the intensity of GFAP-positive expression,and an increase in the level of IL-6,IL-1 β,and TNF-α and the p-JAK2/JAK2 and p-STAT3/STAT3 values(all P＜0.05).Conclusion The GPR110 ligand could alleviate retinal neuroinflammation in diabetic mice by inhibiting the JAK2/STAT3 signaling pathway.

Objective To investigate the mechanism of serine protease inhibitor A3N(SerpinA3N)in alleviating reti-nal neural injury in diabetic mice by inhibiting Müller cell inflammation.Methods Thirty-six db/db mice(72 eyes)were randomly divided into the db/db group,the db/db+SerpinA3N-overexpressing adeno-associated virus(AAV-SerpinA3N)group,and the db/db+empty vector adeno-associated virus(NC-SerpinA3N)group,with 12 mice in each group.Twelve age-matched healthy male littermate mice were randomly selected as the healthy control group(db/m group).Mice in each group were sacrificed 4 weeks after the corresponding treatments.Immunofluorescence staining was used to detect the co-localization of SerpinA3N and glial fibrillary acidic protein(GFAP)in the mouse retina.Hematoxylin-eosin(HE)staining was used to observe histopathological changes in the retinal tissue.TUNEL staining was used to detect the apoptosis of reti-nal ganglion cells(RGCs).Immunohistochemistry was used to detect GFAP expression.ELISA was used to measure the levels of inflammatory factors[interleukin(IL)-1 β,IL-6,IL-18,and tumor necrosis factor-α(TNF-α)]in the retinal tis-sue.The predicted target genes of SerpinA3N were imported into the STRING database to construct a protein-protein inter-action(PPI)network.The highest-scoring target was selected based on the scores for molecular docking.Western blot was used to detect the expression levels of SerpinA3N,nuclear factor kappa B(NF-κB),and spleen focus-forming virus proviral integration oncogene(Spi1)proteins in the retinal tissue.Results Immunofluorescence staining showed co-lo-calized expression of SerpinA3N and GFAP in the retinal tissue.Compared with the db/m group,the db/db,db/db+NC-SerpinA3N,and db/db+AAV-SerpinA3N groups showed decreased retinal thickness and RGC count,and increased number of TUNEL-positive cells,relative GFAP-positive expression intensity,levels of all inflammatory factors,and expression lev-els of NF-κB and Spi1 proteins,while SerpinA3N protein expression was decreased(all P＜0.05).Compared with the db/db group,the db/db+AAV-SerpinA3N group showed increased retinal thickness and RGC count,and decreased number of TUNEL-positive cells,relative GFAP-positive expression intensity,levels of all inflammatory factors,and expression levels of NF-κB and Spi1 proteins,while SerpinA3N protein expression was increased(all P＜0.05).Compared with the db/db+AAV-SerpinA3N group,the db/db+NC-SerpinA3N group showed decreased retinal thickness and RGC count,and increased number of TUNEL-positive cells,relative GFAP-positive expression intensity,levels of all inflammatory factors,and ex-pression levels of NF-κB and Spi1 proteins,while SerpinA3N protein expression was decreased(all P＜0.05).The PPI re-sults indicated an interaction between SerpinA3N and Spi1.Molecular docking results showed that Spi1 could form hydro-gen bonds with residues surrounding SerpinA3N.Conclusion Overexpression of SerpinA3N can inhibit Müller cell in-flammation and ameliorate retinal neural injury in diabetic mice,and the mechanism may be associated with the inhibition of the Spi1/NF-κB signaling pathway.

[Objective]To explore the protective effect of glucocorticoid-induced transcription factor 1(GLCCI1)on cognitive dysfunction in diabetic mice and its mechanism.[Methods]Twenty-four C57BL/6J mice were randomly divided into 4 groups,namely Control,DM,DM+AAV-Glcci1,and DM+AAV-NC.The Control group was intraperitoneally injected with saline,while the other groups were all injected with streptozotocin(STZ).Two weeks after successful modeling,the DM+AAV-Glcci1 group was brain stereotactic injected with Glcci1 overexpressing adeno-associated virus,and the DM+AAV-NC group was stereoscopically injected with the control virus.After 12 weeks,the Morris water maze test was used to evaluate the learning and memory abilities of mice in each group.Subsequently,the localized expression of GLCCI1 in the hippocampus were determined by immunofluorescence and immunohistochemistry experiments.The myelin morphology in the hippocampus was observed by LFB staining,the neuronal morphology was observed by Nissl staining,and the myelin-related proteins MBP and CNPase were stained by immunohistochemistry.Molecular docking was used to predict the interaction between GLCCI1 and HSPA5.The expression of endoplasmic reticulum stress-related proteins was detected by Western blot.[Results]The results of the behavioral experiment showed that compared with the mice in the Control group,DM mice exhibited obvious cognitive dysfunction behaviors(P＜0.000 1),and the learning and memory abilities of mice improved after overexpression of Glcci1(P=0.000 7).The results of immunofluorescence and immunohistochemistry showed that GLCCI1 was expressed in hippocampal neuron cells.Compared with Control mice,the expression level of GLCCI1 in DM mice was significantly downregulated(P＜0.000 1).The molecular docking results revealed that GLCCI1 interacts with HSPA5.The Western blot results indicated that,compared with the Control group,the expression levels of endoplasmic reticulum stress-related proteins HSPA5(P＜0.000 1),ATF4(P＜0.000 1),ATF6(P=0.001 1),and p-ELF2α/elF2α(P=0.000 1)in the DM group were significantly increased;Compared with the DM group,the expression of the corresponding protein HSPA5(P＜0.000 1),ATF4(P＜0.000 1),ATF6(P=0.000 2),and p-ELF2α/elF2α(P=0.000 1)was significantly down-regulated after overexpression of Glcci1.LFB staining showed that compared with the Control group,the myelin integrity of DM mice decreased significantly(P=0.010 3),the expressions of myelin-related proteins MBP and CNPase decreased significantly(P=0.000 4,P=0.000 2),and Nissl staining observed disordered neuronal arrangement.Compared with the mice in the DM group,the myelin integrity in the hippocampal region significantly increased after overexpression of Glcci1(P=0.000 3),the expressions of myelin-related proteins MBP and CNPase significantly increased(P=0.001 4,P=0.000 1),and the ordered arrangement of neurons was observed by Nissl staining.[Conclusion]The down-regulation of GLCCI1 expression in hippocampal neurons promotes demyelination of hippocampal neurons and thereby induces diabetic cognitive dysfunction.The specific mechanism may be related to endoplasmic reticulum stress.

Objective To study the relationship between the changes of TRIM16 in hippocampus of type 2 diabetic rats induced by streptozotocin (STZ) and cognitive dysfunction and apoptosis of hippocampal cells.Methods The male Sprague-Dawley (SD) rats of 8 weeks old were randomly divided into diabetic group (DM group) (n=25) and normal control group (NC group) (n=15).NC group were given normal feed, DM rats were fed with high glucose and high fat diet,followed by low dose of STZ (25 mg/kg, intraperitoneal injection), induced compensatory secretion of insulin, five days after the detection of tail vein blood glucose>16.7 mmol/L, and established the STZ.Meanwhile, citric acid buffer was injected to rats in group as control.After successful modeling, the rats in the two groups were fed with 17 weeks.Morris water maze test was used to detect the cognitive function of the two groups of rats.The protein expression of TRIM16, Bax and Bcl-2 protein in fresh hippocampus tissue of DM group and NC group rats was detected using western blotting.Rats in DM group and NC group were perfused with brain, and the hippocampal tissues were stained with immunohistochemistry.TUNEL method was used to detect the apoptosis of hippocampus cells.ResultsCompared with NC group, morris water maze test showed that the escape latency of rats in DM group is longer than NC group, staying at the original platform quadrant time in DM group is less than the NC group.TUNEL assay showed that after injection of 17 weeks, hippocampal CA1 pyramidal cells in type 2 diabetic rats appeared obvious apoptosis, positive neurons in the nuclei were brown, karyopyknosis anachromasis, while control group of neurons in the nucleus without brown coloring.Compared with the NC group, hippocampus immunohistochemistry results showed that the protein of TRIM16 in hippocampus CA1 region of type 2 diabetic rats was increased, cytoplasm brown, coarse granular.Compared with the NC group, the expression of Bcl-2 had no obvious change in hippocampus of type 2 diabetic rats at 17 weeks, while the expression of TRIM16, Bax and the ratio of Bax/Bcl-2 was significantly increased (P<0.05).Conclusion The protein expression of TRIM16 using immunohistochemical and Western blotting in hippocampus of diabetic rats induced by STZ was increased.There is apoptosis in hippocampus of diabetic rats induced by STZ, the expression of Bcl-2 was not significantly changed, the expression of Bax and the ratio of Bax/Bcl-2 were significantly increased.TRIM16 is involved in diabetic cognitive dysfunction by promoting apoptosis of diabetic rat hippocampus.

OBJECTIVE To clarify the influence factors of hearing impairment in mice caused by lack of folic acid and the mechanism.METHODS Mice were randomly divided into 2 groups,fed with no folic acid diet and normal diet for 10 weeks.Hearing was tested by ABR.Serum homocysteine,folic acid were detected.Immune imprint method was used to analyze the expression of related protein.Cochlear tissue was tested by histological method to observe morphological changes.At the same time using the TUNEL method was used to analyse the cochlear tissue cell apoptosis.RESULTS Analysis showed that serum folate levels in mice fed without folic acid diet dropped than that of the normal group,while homocysteine levels elevated.Through the brain stem auditory records and cochlear cell apoptosis of mice severe hearing loss was found in folate deficiency group.Western blot detection showed enzyme catalysis homocysteine products increased by 50％ in folate deficiency group than that of the normal diet group.CONCLUSION The high homocysteine levels,and folate deficiency can cause hearing loss in mice and are related with the inner ear homocysteine metabolic disorders.

Objective To explore protective effects of panaxtrial saponins (PTS) on cognition and memory of diabetic rats and to reveal its mechanism by which might involve regulating activity of astrocytes. Methods SD rats (n=24) were ran?domly assigned into control, diabetic and PTS-treated groups (n=8 in each group). Rat diabetic model was induced through streptozotocin injection intraperitoneally. Rats in control group were native rats, and rats in PTS-treated group were diabetic rats that were administered with PTS. Body weight and blood glucose were monitored through the experiments. Three months later, state of cognition was examined by methods of water maze. Hippocampal astrocyte morphology were detected by immu?nohistochemistry, and the expression of glial cell line-derived neurotrophic factor (GDNF) and glial fibrillary acidic protein (GFAP) in hippocampus were revealed by Western blot. Results Compared with control group, diabetic group showed cog?nitive dysfunction, atrophic astrocyte soma, shrinked astrocyte processes, and down-regulation of hippocampal GFAP and GDNF (P<0.05). Compared with diabetic group, PTS-treated group exhibited improved cognition and morphology of hippo?campal astrocyte, and reversed expression of GFAP and GDNF in diabetic hippocampus (P<0.05). Conclusion PTS re?versed astrocytic reactivity as well as expression of GDNF and GFAP in diabetic hippocampus and ameliorated diabetic cog?nitive dysfunction.