Chemical composition and protective effect of cerebrospinal fluid of Dan-Deng-Tong-Nao capsules on brain microvascular endothelial cells injured by OGD/R

Ethnopharmacological relevance: Dan-Deng-Tong-Nao Capsules (DDTNC) is a Chinese patent medicine and has been used in treating cerebral ischemic stroke (IS) for a long time in China, protection of brain microvascular endothelial cells (BMECs) is the main treatment strategy. But the holistic chemical information and potential bioactive components of DDTNC on protecting BMECs and its underlying mechanism is still unclear. Aim of the study: To identify the active ingredients of DDTNC and to explore the protective effects of DDTNC on BMECs associated with Wnt/β-catenin pathway. Materials and methods: The components of DDTNC and cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF) were detected by High performance liquid chromatography combined with Diode array detector (HPLC-DAD) and Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), respectively. The experiment rat model was established with middle cerebral artery occlusion (MCAO), the therapeutic effects of DDTNC were assessed by Longa assay and TTC staining. The cerebral micro vessel density was determined by immunofluorescence staining. The injured BMECs caused by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of cerebrospinal fluid containing composition of DDTNC (DDTNC-CSF). The cell survival rate was detected by the method of CCK-8, the intracellular Ca²⁺ and reactive oxygen species (ROS) was estimated by Fluo-3. Moreover, the proteins of Bax, Bcl-2, Wnt, β-catenin, GSK-3β was determined by Western blotting. Results: The RSD values of all methodological studies were less than 3.0%. A total of 20 compounds were detected under the optimized HPLC-DAD chromatographic condition. In the UPLC-Q-TOF-MS negative mode, peak 1 and peak 2 were detecteted in DDTNC-CSF and was identified as Danshensu and Puerarin, respectively. In the UPLC-Q-TOF-MS positive mode, peak 1 and peak 3 were detecteted in DDTNC-CSF and was identified as Danshensu and Scutellarin, respectively. DDTNC significantly decreased the Longa values and infarct volume and significantly increased the cerebral microvessel density of the MCAO rats. The accumulation of intracellular Ca²⁺ and ROS in BMECs injured by OGD/R decreased significantly in DDTNC-CSF group. The expression of Bcl-2, β-catenin, wnt-1 was upregulated by DDTNC-CSF and the level of Bax and GSK3β could be downregulated by DDTNC-CSF. Conclusion: The present study provided a scientific basis for revealing the mechanism of DDTNC in the treatment of IS and DDTNC is expected to be an effective drug for the treatment of IS.