ABSTRACT
Objective:To investigate the protective effect of asiatic acid (AA) on blood-retinal barrier (BRB) in diabetic rats and its possible mechanism.Methods:Ninety-six healthy 8-week-old male SD rats were randomly divided into normal control group, diabetes group, low-dose AA group and high-dose AA group, with 24 rats in each group.Intraperitoneal injection of streptozocin (STZ) was used to establish diabetes model.One month after the establishment of the model, the low-dose AA group and the high-dose AA group were given intragastrical administration of 37.5 mg/kg AA and 75.0 mg/kg AA, respectively, once a day according to grouping.The normal control group and the diabetes group were administrated with the same amount of 0.5% sodium carboxymethyl cellulose.The body weight of the rats were weighted at week 0, 1, 2, 3, 4 after intragastrical administration.Blood was taken from the tail vein and the blood glucose level was measured.The retina was obtained one month following the administration.Pathological changes of the rats retina were detected by hematoxylin-eosin (HE) staining.Evan's blue quantitative method was used to detect the damage of blood-retinal barrier (BRB). Immunofluorescence staining was performed to detect the distribution of Occludin, Notch1, Jagged canonical Notch ligand 1 (JAG1) and Delta like canonical Notch ligand 4 (DLL4) in retina.The mRNA and protein expressive levels of Occludin, Notch1, JAG1 and DLL4 were detected by Real-time PCR and Western blot.The study protocol was approved by a Scientific Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2020-KY-228). The use and care of animals complied with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health and the 3R rules.Results:At 4 weeks after intragastrical administration, the body weight of the high-dose AA group was significantly higher than that of the diabetes group, and the blood glucose values were significantly lower in the high-dose AA group and the low-dose AA group in comparison with the diabetes group (all at P<0.05). The cells were arranged orderly with clear layered structure in the normal control group.In the diabetes group, the retina was thicker than that of the normal control group, with a thicker outer nuclear layer, disordered cell arrangement and unclear layered structure.Compared with the diabetes group, the total retinal thickness and structure were obviously improved in the low-dose AA group and the high-dose AA group.Evan's blue leakage in retina was (3.07±1.30), (13.73±3.88), (9.57±2.69) and (6.55±1.61)ng/mg in the normal control group, the diabetes group, the low-dose AA group and the high-dose AA group, respectively.There was a significant difference in leakage of Evan's blue among the four groups ( F=18.50, P<0.01), among which the leakage of Evan's blue dye in the high-dose AA group was significantly lower than that of the diabetes group ( P<0.01). Compared with the diabetes group, there was significantly higher relative expression level of Occludin protein and significantly lower relative expression levels of Notch1, JAG1 and DLL4 proteins in the other three groups (all at P<0.05). The relative expression level of Occludin protein was significantly higher and the relative expression levels of Notch1, JAG1 and DLL4 proteins were significantly lower in the high-dose AA group than those in the low-dose AA group (all at P<0.05). Compared with the normal control group, the Occludin mRNA expression level was significantly decreased and the expression levels of Notch1, JAG1 and DLL4 mRNA were significantly increased in the diabetes group and low-dose AA group (all at P<0.01). The Occludin mRNA expression level was higher and the Notch1 mRNA expression level was lower in the high-dose AA group than those in the diabetes group and the low-dose AA group, and the expression levels of JAG1 and DLL4 mRNA were lower in the high-dose AA group in comparison with the diabetes group, and the differences were statistically significant (all at P<0.05). Conclusions:Asiatic acid might play a protective role on BRB in diabetic rats by inhibiting Notch1 signaling pathway.
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Objective To investigate the effect of chaihu-shugan-san ( CSS) on the behaviors and Notch1 signal pathway in depression model rats. Methods Thirty-two SD rats with similar behavioral scores were divided into control group (CON),model group (CUMS),positive control group (FLU) and interven-tion group (CSS).The depression model was established by stimulating with chronic unpredictable mild stress (CUMS),and the behaviors evaluation was assessed by sugar water consumption and forced depression.Im-munofluorescence was used to detect the proliferation of hippocampus neurons in rats,at the same time,real- time PCR and Western blot were used to detect the mRNA and protein expression levels of each factor (Notch1,Hes1,Hes5 and Jagged1) of Notch1 signal pathway respectively. Results Compared with CON group,the percentage of sugar water preference and swimming length of rats decreased significantly in CUMS group (P＜0.05 and P＜0.01).Compared with CUMS group,the percentage of sugar water preference and swimming length of rats increased significantly in CSS group(P＜0.05).Compared with CON group,there was a significant increase in the inactivity length of rats between CUMS group,FLU group and CSS group,and the difference was statistically significant (P＜0.01).Compared with CUMS group,the swimming length of rats in CSS group was significantly reduced,and the difference was statistically significant (P＜0.01).Compared with CON group((750.00±27.51)/mm2),the number of BrdU positive cells in the substratum or granulocyte lay-er of the hippocampus dentate gyrus of rats in CUMS group ((338.75±29.61)/mm2),FLU group ((545.00 ±17.73)/mm2) and CSS group ((529.38±13.74)/mm2) was significantly reduced(P＜0.01).Compared with CUMS group ((338.75±29.61)/mm2),there was a significant increase in the number of BrdU positive cells in the substratum or granulocyte layer of the hippocampus dentate gyrus of rats in CSS group ((529.38 ±13.74)/mm2),and the difference was statistically significant (P＜0.01).Compared with CON group,the mRNA and protein expression levels of Notch1,Hes1,Hes5,and Jagged1 in the hippocampus of rats in CUMS group were significantly reduced(P＜0.05 or P＜0.01).Compared with CUMS group,the mRNA and protein ex-pression levels of Notch1,Hes1,Hes5 and Jagged1 in the hippocampus of rats in FLU group and CSS group were significantly increased,and the differences were statistically significant (P＜0.05 or P＜0.01). Conclu-sion Notch1 signal pathway may be related to the obstacle during the hippocampus nerve regenerating in the model rat under chronic unpredictable mild stress.CSS may play an anti-depressant role by regulating Notchl to improve hippocampus nerve regeneration.
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Objective To explore the effect of Notch1 signal pathway on the osteogenic differentiation in human periodontal ligament stem cells(PDLSCs) under dynamic strain.Methods PDLSCs were separated from freshly extracted teeth then identified and prolifed.Notch1 signal pathway was regulated by chemicals.Dynamic strains were applied to PDLSCs with the tension plus system.Then Notch1 signal pathway key factor Notch intracellular domain(NICD),osteoblastic related indexes alkaline phosphatase(ALP) and bone morphogenetic proteins 2 (BMP2) were detected by western blot expression.The deformation rate of stress parameters was 0 to 12%,and the frequency was 0.1 Hz.The loading time was 0 h,6 h,12 h and 24 h.Results As Notch1 signal pathway was activated,the expression of osteogenic markers ALP and BMP2 both reduced (P<0.05).On the contrary, the expression of osteogenic markers ALP and BMP2 both increased obviously (P<0.05) as Notch1 signal pathway was inhibited.Conclusion The activated Notch1 signal pathway will inhibit osteogenic differentiation of PDLSCs under dynamic tensile.