Protective Effect Of Daming Capsule Against Vascular Dementia In Rats And Its Possible Mechanism

Background and PurposeVascular dementia?VD?is an acquired syndrome of step-wise deterioration in intellectual powers caused by cerebral vascular diseases with the most common reason of chronic cerebral ischemia?CCI?.Along with the population aging,its incidence rate increases year by year.It is life-threatening and affecs life quality of patients and aggravates the burden of whole the society and family.Therefore,to investigate the safe and effective anti-VD drugs has become a vital research topic.In the present study,we aim to predict the protective effect of Daming Capsule?DMC?against VD induced by CCI by a network pharmacology method,and speculate the possible mechanisms,then validate and evaluate its protective effect and mechanisms by in vivo and in vitro experiments.The study could provide theoretical and experimental bases for the expansion of clinical indications of DMC and the prevention and control of VD.Methods1.Constituent information about each herb of DMC was obtained from the TCM Database @Taiwan database.After that,the STITCH 3.1 database was used to search for corresponding targets to each constituent,the Hu GE Navigator database was applied to obtain experimentally validated CCI-related genes.Herb-constituent-CCI-specific targets association networks were established by Cytoscape.The potential anti-VD action by DMC was forecasted by the results of the networks and the possible mechanisms were suggested.2.In vitro experiment,PC12 cell was induced by H₂O₂ and used to mimic the effect of oxidative stress on VD.The method of serum pharmacology was used to investigate the protective effect of DMC-containing serum on H₂O₂-induced PC12 cell injury.Rats were randomly divided into four groups: high-dose DMC-containing serum?600 mg/kg?,moderate-dose DMC-containing serum?300 mg/kg?,low-dose DMC-containing serum?150mg/kg?and control group?physiological saline?.Serum was collected after gavage for 3 d.Fistly,to investigate the protective effect of DMC on H₂O₂-induced PC12 cell injury,methyl thiazolyl tetrazolium?MTT?method and lactate dehydrogenase?LDH?assay was used to detect the effects of DMC-containing serum on cell viability and investigate the effects of DMC-containing serum on cell membrane structures.Then,to investigate the protective mechanism of DMC on H₂O₂-induced PC12 cell injury,the SOD activity and MDA level were assayed to evaluate the anti-oxidative ability of DMC-containing serum.3.In vivo experiment,rats with permanent bilateral occlusion of carotid arteries?2-VO?were used to mimic VD induced by CCI.All the rats were randomly divided into five groups: sham-operated group?physiological saline?,2-VO group?physiological saline?,low dose of DMC group?100 mg/kg?,high dose of DMC group?200 mg/kg?and positive control medicine hydergine group?0.6 mg/kg?.Fistly,spatial learning and memory abilities were evaluated by Morris water maze,and morphological changes of cerebral cortex and hippocampus were assessed using hematoxylin and eosin staining?HE?.To detecte the level of oxidative stress in brain tissue,malondialdehyde?MDA?levels,superoxide dismutase?SOD?activity,glutathione peroxidase?GSH-PX?activity and catalase?CAT?activity were measured by biochemical analysis.To detecte the level of inflammation in brain tissue,inflammatory markers?TNF-a,IL-6 and IL-1??were measured by enzymelinked immunosorbent assay?ELISA?.Evans blue?EB?staining method was used to evaluate the blood brain barrier permeability change.Results1.The network pharmacology results showed that 28 constituents of DMC targeted 24 targets related to CCI and 59 target-constituent associations were identified.So DMC may show protective effect against VD induced by CCI.By the network pharmacology analysis and investigation literature,we speculated that the protection mechanisms are most likely related,at least in part,its anti-oxidant action,anti-inflammatory action and the protective function on blood brain barrier.2.Expose of PC12 cells to H₂O₂ induced cell injury.DMC-containing serum could improve the viability of PC12 cells,decreased the leakage of LDH,reduced the production of lipid peroxide and increased the activity of SOD.3.Behavior experiment?morris water maze?showed that the escape latency of the DMC?200 mg/kg?group significantly decreased on the fifth day than that of 2-VO group,similar to that of the hydergine group.In the probe trials,the percentage of swimming distance in the target quadrant and the times through the platform of the DMC?200 mg/kg?group was significantly increased compared to that in the 2-VO group.4.HE staining proved that the abnormal morphology in hippocampal CA1 area and cerebral cortex was relieved in DMC?200 mg/kg?group,DMC?100 mg/kg?group and hydergine group compared to that in the 2-VO group,especially DMC?200 mg/kg?group and hydergine group.5.Biochemical assays?SOD,MDA,GSH-Px and CAT?also revealed that the oxidative stress response was induced by CCI.200 mg/kg DMC decreased MDA levels and CCI-elevated SOD activities,and increased the activities of GSH-PX and CAT.6.Elisa assays revealed that the inflammatory response was induced by CCI.TNF-a,IL-6 and IL-1?levels in DMC?200 mg/kg?group and hydergine group were reduced significantly compared to 2-VO group,and IL-6 levels in DMC?100 mg/kg?group were also reduced significantly compared to 2-VO group.7.The effect of DMC on the permeability of blood brain barrier in 2-VO rats showed that 200 mg/kg DMC could improve the change of blood brain barrier of 2-VO rats.ConclusionsThe potential anti-VD action by DMC was forecasted by the results of the network pharmacology and the possible mechanism was suggested.Then the action and possible mechanism were validated by in vivo and in vitro experiments.The in vitro experiment demonstrated that DMC has protective effect on H₂O₂-induced PC12 cell injury,and its mechanism is related to its antioxidant effect.The in vivo experiment demonstrated that DMC improves CCI induced cognitive impairment and attenuates neuron injuries,which are most likely related,at least in part,its antioxidant action,anti-inflammatory action and the protective function on blood brain barrier.