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Objective:To investigate the effect of heat shock protein B8 (HspB8) downregulation on retinal ganglion cell (RGC) and retinal function in the mice model of optic nerve injury (ONC).Methods:Adeno-Associated Virus (AAV) 2 AAV2-shHspB8-GFP was constructed to knockdown HspB8. 66 adult male C57/BL6 mice were randomly divided into the control group, the ONC group, the AAV2-shHspB8 group, the ONC+AAV2-shHspB8 group, and the ONC+AAV2-GFP group. There were 10, 20, 16, 10 and 10 mice respectively, and both eyes were used as experimental eyes. Western blot was used to evaluate the expression of HspB8 on day 3 and 7 after ONC. By GFP immunofluorescence staining, the efficacy of AAV2-shHspB8-GFP transfer was accessed. Moreover, it was possible to identify functional and RGC survival differences between groups by optomotor response (OMR), dark adapted full-field flash electroretinogram (ff-ERG), oscillatory potentials (OPs), photopic negative response (PhNR) and retinal flat-mount RGC counting 5 days after ONC. Comparisons between two groups were made using Mann-Whitney U test, unpaired t-test, unpaired t-test with Welch’s correction, one-way ANOVA, and Bonferroni t test. Results:Compared with the control group, the expression of HSPB8 protein in the retina of mice in ONC3 group was significantly increased, and the difference was statistically significant ( F=43.63, P<0.01). Compared with the control group, the ONC group showed obviously lower visual acuity ( P<0.01), lower a-wave, b-wave, OPs, PhNR amplitude, longer b-wave latency ( P<0.05), and the survival rates of RGC in ONC3 group, ONC5 group and ONC7 group decreased in a time-dependent manner( F=384.90, P<0.01). Transfection of AAV2 efficiency was highest on 4 weeks after IVT. Besides, there was no significant differences between the control group and the AAV2-shHspB8 group on visual acuity, ff-ERG, OPs, PhNR and RGC survival ( P>0.05). In comparison of the control group, we found that RGC survival of the ONC5+AAV2-shHspB8 group was significantly elevated ( F=10.62, P<0.01). Conclusions:Expression of HspB8 on the retina can be induced by ONC. The investigation of RGC counting, visual acuity, and ff-ERG revealed that optic nerve injury destructed functionality of mice retina and resulted to RGC death ultimately. The Most crucial finding of this research is that HspB8 knockdown had a neuroprotective effect in RGC after ONC.
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Objective:To investigate the effects of Krüppel-like factor 7 (KLF7) on the survival of retinal ganglion cells (RGCs) and electroretinogram (ERG) after retinal ischemia-reperfusion (RIR) injury in mice.Methods:A total of 126 male C57BL/6J mice were randomly divided into normal group, RIR group, normal-KLF7 group, normal-green fluorescent protein (GFP) group, RIR-KLF7 group and RIR-GFP group. At the age of 8 weeks, mice of normal-KLF7 group and RIR-KLF7 group were intravitreally injected 1ul of 1.0×10 12 vg/ml adeno-associated virus overexpressing KLF7 (AAV2-KLF7-GFP). Mice of normal-GFP group and RIR-GFP group were injected adeno-associated virus of AAV2-GFP with the same titer. At the age of 11 weeks, RIR injury was induced in mice of RIR group, RIR-KLF7 group and RIR-GFP group, and intraocular pressure was measured. Retinal cryosections were used to access the efficacy of virus transfection 4 weeks after AAV2-KLF7-GFP transfer. 7 days after RIR injury, RGCs' survival rate was observed and quantified by immunofluorescent staining. ERG was performed to observe the differences in amplitudes and incubation period of scotopic ERG a-, b-wave, oscillatory potentials (Ops), photopic negative responses (PhNR). Optomotor response was performed to observe the differences of visual acuity. Expression of KLF7 was detected by western blot 4 weeks after AAV2-KLF7-GFP transfer. Results:Compared with normal group, RGCs’ survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR and visual acuity of mice in RIR group were decreased, and the differences were statistically significant ( t=12.860, 7.157, 5.735, 8.953, 4.744, 9.887; P<0.05). With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. Compared with RIR group, RGCs’ survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR and visual acuity of mice in RIR-KLF7 group were increased, and the differences were statistically significant ( t=6.350, 3.253, 3.695, 5.825, 5.325, 4.591; P<0.05). Protein level of KLF7 was up-regulated in normal-KLF7 group than those in normal group, and the difference was statistically significant ( t=4.105, P<0.01). Conclusion:Overexpression of KLF7 can improve RGCs’ survival rates and preserve the electrophysiological function.
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Objective:To investigate the expression of integrin-linked kinase (ILK)/zinc finger transcription factor (Snail) signaling pathway in renal tubular epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN) and the effect of rhein.Methods:Healthy male Wistar rats of 8 weeks old were randomly divided into normal group, diabetic nephropathy group, rhein intervention group and valsartan intervention group, with 12 rats in each group. Streptozotocin (STZ) was used to induce the diabetic nephropathy model, then rhein intervention group and valsartan intervention group were given rhein 100 mg/(kg·d) and valsartan 30 mg/(kg·d), respectively. At the end of the 8th and 16th week, six rats of each group were killed, in situ lavage kidney, take out the kidney tissue of rats after fixed in wax block and slices. Renal tubular interstitial damage index and the relative area of interstitial collagen evaluated by hematoxylin-eosin (HE) and Masson staining respectively. The protein expression of E-cadherin, α-smooth muscle actin (α-SMA), ILK, Snail and matrix metalloproteinase-9 (MMP-2) in renal tubular epithelial cells were detected by immunohistochemistry.Results:Comparing to normal group, the renal tubular interstitial damage index and relative area of renal interstitial collagen of diabetic nephropathy rats were both increased. The expression of E-cadherin in renal tubular epithelial cells decreased and the expression of α-SMA significantly increased ( P<0.05). Comparing with diabetic nephropathy group, in rhein and valsartan intervention groups, the expression of E-cadherin in renal tubular epithelial cells increased, while the expression of α-SMA significantly decreased ( P<0.05). There was no significant difference in the above indexes between rhein intervention group and valsartan intervention group ( P>0.05). Compared to normal group, the expressions of ILK, Snail, MMP-2 increased progressively with the disease ( P<0.05). Compared with diabetic nephropathy group, rhein and valsartan intervention groups showed significant decrease in expression of ILK, Snail, MMP-2 ( P<0.05). There was no significant difference in the above indexes between rhein intervention group and valsartan intervention group ( P>0.05). Conclusions:Rhein could inhibit EMT progression by down-regulating the expression of ILK/Snail signaling pathway in renal tubular epithelial cells of DN rats.
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@#Objective To observe effects of the electroacupuncture combined with music therapy on cerebral neurometabolites measured with magnetic resonance spectroscopy in patients with post-stroke depression.Methods From January, 2013 to December, 2017, 60 patients with post-stroke depression were randomly divided into drug group (n=30) and electroacupuncture music group (n=30). Another 20 cases of normal control group were recruited. The concentration of N-acetylaspartate (NAA), creatine (Cr) and choline (Cho) in the bilateral prefrontal white matter was measured with magnetic resonance spectroscopy before and after eight-week treatment in three groups. The clinical efficiency was defined as the different of Hamilton Depression Rating Scale before and after treatment.Results There was no significant difference in the effective rate between two treatment groups (Z=-0.145, P>0.05). Before treatment, the Cho/Cr was lower in the electroacupuncture music group and the drug group than in the normal control group (t>3.093, P<0.05). After treatment, the Cho/Cr increased in both treatment groups (t>2.219, P<0.05), and no significant difference was found between them (P>0.05).Conclusion The choline level may be the neurobiochemical basis for glial cell or myelin sheath function and integrity in white matter of patients with post- stroke depression. The regulation of glial cell and myelin function in prefrontal white matter may be one of the action targets of electroacupuncture combined with music therapy.
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Objective To investigate the possible regulating effect of integrin-linked kinase (ILK) towards matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 (MMP-9/TIMP-1) ratio in the process of transforming growth factor-β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial (HK-2) cells.Methods HK-2 cells were cultured and stimulated with 10 ng/ml TGF-β1.Specific ILK-small interfering RNA (ILK-siRNA) was used to inhibit ILK expression.The characteristic epithelial marker (E-cadherin) and mesenchymal marker (α-SMA) of EMT were examined by Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and Western blot.The expressions of ILK,MMP-9,and TIMP-1 were also examined,to determine the regulating effect of ILK towards MMP-9/TIMP-1 ratio.Results In the HK-2 cells cultured with TGF-β1,the expression of E-cadherin decreased,and α-SMA expression increased;overexpression of ILK and an abnormal changing of MMP-9/TIMP-1 ratio were observed.ILK inhibition by ILK-siRNA could adjust MMP-9/TIMP-1 ratio to near normal.Meanwhile,the overexpressed ILK and α-SMA were decreased.Conclusions Our data indicates that ILK-siRNA successfully inhibits ILK expression,which regulates the MMP-9/TIMP-1 ratio in HK-2 cells.The inhibition of ILK expression suppresses TGF-β1-induced EMT partially.