Metabolomics reveals that Da Chuanxiong Formula ameliorates cerebral ischemia/reperfusion injury via regulating membrane phospholipid homeostasis

BACKGROUND: Ischemic stroke remains a major disease leading high disability and mortality worldwide, however, even after timely thrombolytic therapy, the secondary brain injuries induced by cerebral ischemia-reperfusion still cannot be ignored. After cerebral ischemia/reperfusion, abnormal membrane lipids metabolism usually occurred in the brain, which further aggravated the brain injuries and neurological dysfunction. Da ChuanXiong (DCX) Formula, a classic prescription composed of Gastrodia elata Bl. and Ligusticum chuanxiong Hort., exhibits considerable promise in treating ischemic cerebrovascular diseases. This study aims to explore whether DCX could improve cerebral ischemia/reperfusion injury (CIRI) by reshaping membrane lipids homeostasis as well as illustrating the relevant molecular mechanisms. METHODS: Firstly, DCX extract was delivered to middle cerebral artery occlusion-reperfusion (MCAO/R) rats, and neurological function scores, TTC staining and oxidative stress kit assays were conducted to evaluate the neuroprotective effects of DCX. Then, the integrated metabolomics studies of plasma and brain tissue as well as the construction of metabolic network were performed to further detect the alterations of metabolites, unearth the key targets and signaling pathways related to membrane lipid metabolism. Finally, the molecular biological techniques such as real-time PCR and western blot were employed to verify the expression levels of key targets associated with glycerophospholipid metabolism. RESULTS: After CIRI, DCX treatment relieved neurological function deficits, reduced the cerebral infarction areas and elevated the levels of Ach in the brain. Additionally, DCX treatment alleviated the oxidative stress damages via elevating the expressions of GSH and SOD while reducing the level of MDA. Metabolomics studies revealed that DCX was able to reverse 34 differential metabolites in plasma and 40 differential metabolites in brain tissues. Notably, phospholipid-related metabolites were dominant among the differential metabolites, and DCX intervention reversed the abnormality of these differential metabolites. For example, DCX treatment obviously elevated the contents of choline and phosphocholine in the brain tissues, upregulated the contents of PC(16:0/18:2) and PC(16:1(9Z)/18:2(9Z,12Z)), and downregulated the contents of LysoPC, such as LysoPC(14:0), LysoPC(15:0), LysoPC(20:4(5Z,8Z,11Z,14Z)), LysoPC(18:2(9Z,12Z)), LysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)) and LysoPC(P-16:0). KEGG enrichment analyses presented that glycerophospholipid metabolism may be the key regulatory pathway. The constructed metabolic network indicated that PLA2G10, PLA2G2A, PLA2G6, ACHE, CHAT, CHPT1, LPCAT1, LCAT, LPGAT1 and PCYT1B were possible to be ten crucial targets by which DCX improved CIRI via modulating membrane phospholipid metabolisms. Finally, PCR and western blot experiments further testified that DCX could significantly upregulate the expression levels of CHAT and PCYT1B, and obviously downregulate the expression levels of ACHE and PLA2G2A. CONCLUSION: DCX played obvious neuroprotective effects against CIRI. Mechanistically, DCX maintained the balance of lipid metabolites in MCAO/R rats, reshaped cell membrane phospholipids and sustained the stability of cell membrane structure directly or indirectly. And ACHE, CHAT, PCYT1B and PLA2G2A were the crucial metabolic enzyme targets of DCX treating CIRI via regulating membrane phospholipid homeostasis. These findings offered new insights into the potential neuroprotective mechanisms of DCX treating CIRI from the perspective of membrane lipid metabolism for the first time.