ABSTRACT
Our previous studies have shown that puerarin, an active component of the traditional Chinese medicine-Pueraria Lobata, can improve glycometabolism in high-fat diet (HFD) mice with diabetes by activating the glucagon-like peptide-1 receptor (GLP-1R) pathway. This study intends to further evaluate the effect of puerarin on depressive symptoms in HFD mice. Long-term HFD induces type 2 diabetes and depressive-like symptoms in mice. Animal welfare and experimental procedures follow the regulations of the Animal Ethics Committee of the Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Traditional Chinese Medicine (approval No. AEWC-025). The experiment was divided into: control group, model group, model/puerarin (150 mg·kg-1·day-1) group, and model/fluoxetine (15 mg·kg-1·day-1) group. The oral glucose tolerance test (OGTT) and behavioral experimental analysis were performed after 6 weeks of continuous administration. Afterwards, enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-1β (IL-1β), interleukin-6 (IL-6), 5-hydroxytryptamine (5-HT), and corticosterone (CORT) in serum of mice for each group. Western blot assays were used to detect the level of activation and expression of proteins related to neuroplasticity and depressive disorder in the hippocampus. Moreover, HT-22 cell line was used to investigate the protective effect of puerarin on cell morphology and survival. The results show that puerarin can effectively maintain the survival of HT22 in an environment with high glucose and corticosterone. Meantime, the glycemic regulation of diabetic mice was improved after treatment of puerarin, the depressive symptoms were alleviated, the 5-HT increased, and the corticosterone, IL-1β, and IL-6 decreased in the serum. The up-regulation of related proteins in GLP-1R/Wnt/mTOR (mammalian target of rapamycin) signaling in hippocampus suggests that its effect on ameliorating depression in diabetic mice may be related to the activation of GLP-1R/Wnt/mTOR signaling pathway. This study shows that puerarin can significantly ameliorate the depressive symptoms of HFD induced diabetic mice which might be achieved through activating the GLP-1R/Wnt/mTOR signaling pathway and improving hippocampal neuroplasticity.
ABSTRACT
OBJECTIVE@#To investigate the expression changes of the epigenetic regulator enhancer of zeste homolog 2 (EZH2) during pulp inflammation and the effect of EZH2 on macrophages migration.@*METHODS@#Rat dental pulp was stimulated with 10 g/L lipopolysaccharide (LPS) to establish a model of rat pulpitis at different stages of inflammation. Immunohistochemical staining was used to detect the expression changes of EZH2 during the progression of pulp inflammation. Immunofluorescence double staining was used to detect the expression of EZH2, CD68 and their colocalization. To screen the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and human leukaemia-derived monocytic cell line (THP-1) cells, the effects of different concentrations (1, 10, 20, 40, and 100 μg/L) of EZH2 recombinant protein on proliferation of human dental pulp cells (hDPCs) and human monocyte cell line THP-1 were detected by cell counting kit-8 (CCK-8). Transwell migration assay was used to detect the effect of supernatants of hDPCs treated with EZH2 recombinant protein on the migration of THP-1 cells.@*RESULTS@#HE staining results showed that in the model of rat pulp inflammation induced by LPS, with the prolongation of LPS stimulation, the inflammation response of pulp gradually increased. Immunohistochemical results showed that EZH2 expression decreased within 8 h of LPS-induced dental pulp inflammation; but after 1, 3, and 7 d of stimulation, EZH2 expression gradually increased with the extension of the stimulation time. As for the normal rat dental pulp tissue, the positive expression of EZH2 was scattered in the odontoblast cell layer and the pulp proper. Compared with the control group, LPS stimulated the expression of EZH2 and CD68 in the infected dental pulp, and the colocalization of EZH2 and CD68 could be detected in macrophages. The results of CCK-8 suggested that the appropriate concentration of EZH2 recombinant protein to stimulate hDPCs and THP-1 cells was 20 μg/L. Transwell cell migration assay confirmed that compared with the supernatant of EZH2 untreated HDPCs group, the supernatant of EZH2treated hDPCs significantly promoted macrophage chemotaxis.@*CONCLUSION@#EZH2 is involved in the development of pulpitis and promotes the chemotaxis of macrophages, which suggests that EZH2 may play an important regulatory role in the development of pulp inflammation.