Abstract
Diabetic cognitive dysfunction is a serious complication of type 2 diabetes mellitus (T2DM), which can cause neurological and microvascular damage in the brain. At present, there is no effective treatment for this complication. Bushen Huoxue prescription (BSHX) is a newly formulated compound Chinese medicine containing 7 components. Previous research indicated that BSHX was neuroprotective against advanced glycosylation end product (AGE)-induced PC12 cell insult; however, the effect of BSHX on AGE-induced cerebral microvascular endothelia injury has not been studied. In the current research, we investigated the protective effects of BSHX on AGE-induced injury in bEnd.3 cells. Our findings revealed that BSHX could effectively protect bEnd.3 cells from apoptosis. Moreover, we analyzed the network regulation effect of BSHX on AGE-induced bEnd.3 cells injury at the proteomic level. The LC-MS/MS-based shotgun proteomics analysis showed BSHX negatively regulated multiple AGE-elicited proteins. Bioinformatics analysis revealed these differential proteins were involved in multiple processes, such as Foxo signaling pathway. Further molecular biology analysis confirmed that BSHX could downregulate the expression of FoxO1/3 protein and inhibit its nuclear transfer and inhibit the expression of downstream apoptotic protein Bim and the activation of caspase, so as to play a protective role in AGE-induced bEnd.3 injury. Taken together, these findings demonstrated the role of BSHX in the management of diabetic cerebral microangiopathy and provide some insights into the proteomics-guided pharmacological mechanism study of traditional Chinese Medicine.
Highlights
Diabetes mellitus (DM) is a common metabolic disease that can be accompanied by a variety of complications
Antibodies against cleaved caspase-3, Bim, Histone H3, FoxO1, FoxO3a, α-Tubulin, and rabbit IgG were obtained from Cell Signaling Technology (Boston, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was bought from Sigma-Aldrich Chemical Co. (Saint Louis, MO, USA). e Dulbecco’s modified eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from PANBiotech GmbH (Aidenbach, Germany). e endothelial cell medium (ECM) was obtained from Beijing M&C Gene Technology Ltd. (Beijing, China). e lactate dehydrogenase (LDH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China)
We found that LDH released in the supernatant of cells from the model group dramatically swelled to 142.02 ± 13.77 U/L (P < 0.001 vs. Control group) and Bushen Huoxue prescription (BSHX) (100 mg/L) significantly shrunk the release and remedied the cytotoxity (74.83 ± 9.28 U/L, P < 0.01 vs. model group)
Summary
Diabetes mellitus (DM) is a common metabolic disease that can be accompanied by a variety of complications. Diabetic cognitive dysfunction is a major central nervous system complication of DM, mainly manifested as cognitive impairment and neurodegeneration [1,2,3]. The pathogenesis of this complication is still not clear, recent studies have found that overproduction of advanced glycosylation end products (AGEs) is closely related to the occurrence of cognitive dysfunction [4, 5]. AGEs are toxic substances produced by nonenzymatic glycosylation of proteins and reducing sugars under long-term hyperglycemia. AGEs can damage cells and even kill them by inducing oxidative stress and reactive oxygen species (ROS) formation [4, 10, 11]. Cerebral microvascular endothelial cells are the basic oxygen supply unit of brains and the blood-brain barrier. In diabetic patients with long-term hyperglycemia, the intense nonenzymatic glycosylation reactions provoke the accumulation
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