Protection against acute cerebral ischemia/reperfusion injury by QiShenYiQi via neuroinflammatory network mobilization.

Cerebral ischemia/reperfusion injury (CI/RI) is a common feature of ischemic stroke, involving a period of impaired blood supply to the brain, followed by the restoration of cerebral perfusion through medical intervention. Although ischemia and reperfusion brain damage is a complex pathological process with an unclear physiological mechanism, more attention is currently focused on the neuroinflammatory response of an ischemia/reperfusion origin, and anti-inflammatory appears to be a potential therapeutic strategy following ischemic stroke. QiShenYiQi (QSYQ), a component-based Chinese medicine with Qi-tonifying and blood-activating property, has pharmacological actions of anti-inflammatory, antioxidant, mitochondrial protectant, anti-apoptosis, and antiplatelet aggregation. We have previously reported that the cardioprotective effect of QSYQ against ischemia/reperfusion injury is via improvement of mitochondrial functional integrity. In this research work, we aimed to investigate the possible mechanism involved in the neuroprotection of QSYQ in mice model of cerebral ischemia/reperfusion injury based on the inflammatory pathway. The cerebral protection was evaluated in the stroke mice after 24 h reperfusion by assessing the neurological deficit, cerebral infarction, brain edema, BBB functionality, and via histopathological assessment. TCM-based network pharmacology method was performed to establish and analyze compound-target-disease & function-pathway network so as to find the possible mechanism linking to the role of QSYQ in CI/RI. In addition, RT-qPCR was used to verify the accuracy of predicted signaling gene expression. As a result, improvement of neurological outcome, reduction of infarct volume and brain edema, a decrease in BBB disruption, and amelioration of histopathological alteration were observed in mice pretreated with QSYQ after experimental stroke surgery. Network pharmacology analysis revealed neuroinflammatory response was associated with the action of QSYQ in CI/RI. RT-qPCR data showed that the mice pretreated with QSYQ could significantly decrease IFNG-γ, IL-6, TNF-α, NF-κB p65, and TLR-4 mRNA levels and increase TGF-β1 mRNA level in the brain compared to the untreated mice after CI/RI (p < 0.05). In conclusion, our study indicated the cerebral protective effect of pretreatment with QSYQ against CI/RI, which may be partly related to its potential to the reduction of neuroinflammatory response in a stroke subject.

Wang Y ，Xiao G ，He S ，Liu X ，Zhu L ，Yang X ，Zhang Y ，Orgah J ，Feng Y ，Wang X ，Zhang B ，Zhu Y ... -  《-》

Synergy of "Yiqi" and "Huoxue" components of QishenYiqi formula in ischemic stroke protection via lysosomal/inflammatory mechanisms.

Ischemic stroke is one of the leading causes of mortality and long-term disability worldwide. Currently, approved therapies of intravenous thrombolysis and mechanical thrombectomy are limited only to selected patients with rescuable brain tissue. Chinese medicine that benefits Qi (Yiqi, YQ) and activates blood (Huoxue, HX) is widely used in the clinic for treating stroke, but their mechanisms are not well understood yet. We have previously reported that QishenYiqi (QSYQ) formula exerts cerebral protective effect and promotes post-stroke recovery. This study aimed to explore the chemical basis and molecular mechanism of anti-stroke therapy of QSYQ and its YQ and HX components further. Serum pharmacochemistry was performed to identify the bioactive constituents in QSYQ for cerebral protection. The survival rate, mNSS test, open field test, gait analysis, cerebral infarction volume, and blood-brain barrier (BBB) integrity were determined to uncover the synergistic and differential contributions of YQ and HX components in a cerebral ischemia/reperfusion injury (CI/RI) model. Bioinformatic mining of QSYQ proteomics data and experimental validation were executed to access the functional mechanism of YQ and HX components. Eleven prototype ingredients and six metabolites were successfully identified or tentatively characterized in rat plasma. Therapeutically, YQ and HX components of QSYQ synergistically boosted the survival rate, improved neurological and motor functions, alleviated cerebral infarction as well as protected BBB integrity in CI/RI model in rats. Individually, YQ component contributed more to ameliorating locomotive ability than that of HX component. Mechanistically, HX component played a more prominent role in the modulation of galectin-3 mediated inflammation whereas YQ component regulated lysosomal-autophagy signaling. This study identifies major prototype ingredients and metabolites of QSYQ in plasma which may contribute to its cerebral protection. YQ and HX components of QSYQ differentially and synergistically protect the brain from CI/RI by regulating galectin-3-mediated inflammation and lysosomal-autophagy signaling. These findings demonstrate that a maximal stroke protection by a component-based Chinese medicine could be attributed to the combination of its individual components via different mechanisms. It may shed new light on our understanding of the TCM principle of tonifying Qi and activating blood, particularly in a setting of ischemic stroke.

Wang Y ，Liu X ，Zhang W ，He S ，Zhang Y ，Orgah J ，Wang Y ，Zhu Y ... -  《-》

Galectin-3 Mediated Inflammatory Response Contributes to Neurological Recovery by QiShenYiQi in Subacute Stroke Model.

Effective therapies for stroke are still limited due to its complex pathological manifestations. QiShenYiQi (QSYQ), a component-based Chinese medicine capable of reducing organ injury caused by ischemia/reperfusion, may offer an alternative option for stroke treatment and post-stroke recovery. Recently, we reported a beneficial effect of QSYQ for acute stroke via modulation of the neuroinflammatory response. However, if QSYQ plays a role in subacute stroke remains unknown. The pharmacological action of QSYQ was investigated in experimental stroke rats which underwent 90 min ischemia and 8 days reperfusion in this study. Neurological and locomotive deficits, cerebral infarction, brain edema, and BBB integrity were assessed. TMT-based quantitative proteomics were performed to identify differentially expressed proteins following QSYQ treatment. Immunohistochemistry, western blot analysis, RT-qPCR, and ELISA were used to validate the proteomics data and to reveal the action mechanisms. Therapeutically, treatment with QSYQ (600 mg/kg) for 7 days significantly improved neurological recovery, attenuated infarct volume and brain edema, and alleviated BBB breakdown in the stroke rats. Bioinformatics analysis indicated that protein galectin-3 and its mediated inflammatory response was closely related to the beneficial effect of QSYQ. Specially, QSYQ (600 mg/kg) markedly downregulated the mRNA and protein expression levels of galectin-3, TNF-α, and IL-6 in CI/RI brain as well as serum levels of TNF-α and IL-6. Overall, our findings showed that the effective action of QSYQ against the subacute phase of CI/RI occurs partly via regulating galectin-3 mediated inflammatory reaction.

Wang Y ，He S ，Liu X ，Li Z ，Zhu L ，Xiao G ，Du X ，Du H ，Zhang W ，Zhang Y ，Orgah J ，Feng Y ，Zhang B ，Zhu Y ... -  《Frontiers in Pharmacology》

Mailuo Shutong pills inhibit neuroinflammation by regulating glucose metabolism disorders to protect mice from cerebral ischemia-reperfusion injury.

Mailuo Shutong Pill (MLST), a traditional Chinese medicine (TCM), has been widely used for clearing heat and detoxifying, eliminating stasis and dredging meridians, dispelling dampness and diminishing swelling. Earlier study found that MLST could improve cerebral ischemic-reperfusion injury, however, the potential mechanism has not been well evaluated. In this study, a well established and widely used mice model of middle cerebral artery occlusion/reperfusion (MCAO/R) was preformed to evaluate the protective function of MLST on cerebral ischemic-reperfusion injury and further discuss the potential pharmacological mechanisms. Chemical profiling of MLST was analyzed based on Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry. ICR mice were challenged by MCAO/R surgery. The protective effect of MLST on MCAO/R injury was evaluated by neurological deficit score, cerebral infarct rate, brain water content, H&E and nissl staining. The blood-brain barrier (BBB) integrity was detected by Evans blue staining. The potential pharmacological mechanism of MLST in treating MCAO/R injury was further elucidated by the methods of proteomics, central carbon targeted metabolomics, as well as Western blot. Immunohistochemistry was used to detect the microglia infiltration, enzyme linked immunosorbent assay (ELISA) kit was explored to evaluate the content of IL-1β, TNF-α and IL-6 in brain tissue, and Western blot was used to detect proteins expression in brain tissue. A total of 76 chemical compounds have been determined in MLST. MLST effectively protected mice from MCAO/R injury, which was confirmed by lower neurological deficit score, cerebral infarct rate, brain water content and nissl body loss, and improved brain pathology. Meanwhile, MLST upregulated the expression of ZO-1, Occludin and Claudin 5 by downregulating the ratio of TIMP1/MMP9 to suppress the entrance of Evans blue to brain tissue, indicating that MLST maintained the integrity of BBB. Further studies indicated that MLST inhibited the inflammatory level of brain tissue by inhibiting microglia infiltration and downregulating NLRP3 inflammasome signaling pathway. The results of proteomics, Western blot, and central carbon targeted metabolomics confirmed that MLST regulated Glycolysis/Gluconogenesis, Pyruvate metabolism and TCA cycle in brain tissue of mice with MCAO/R. MLST inhibits neuroinflammation by regulating glucose metabolism disorders to interfere with immune metabolism reprogramming and inhibit the NLRP3 inflammasome signaling pathway, and finally improve cerebral ischemia-reperfusion injury. This study confirms that MLST is a potential drug for treating Cerebral ischemic stroke.

Guan Y ，Pan L ，Niu D ，Li X ，Li S ，Cheng G ，Zeng Z ，Yue R ，Yao J ，Zhang G ，Sun C ，Yang H ... -  《-》

D-allose protects the blood brain barrier through PPARγ-mediated anti-inflammatory pathway in the mice model of ischemia reperfusion injury.

Our early experiments confirmed that D-allose was closely involved in the blood brain barrier (BBB) protection from ischemia reperfusion (IR) injury, but the regulatory mechanism is not fully defined. In this study, we aimed to investigate the role of D-allose in the protection of BBB integrity and the relevant mechanisms involved in the mice model of middle cerebral artery occlusion and reperfusion (MCAO/Rep). D-allose was intravenously injected via a tail vein (0.2mg/g and 0.4mg/g, 1h before ischemia), GW9662 was intraperitoneal injected to the mice (4mg/kg) before inducing ischemia 24h. Pretreatment with D-allose ameliorated the neurological deficits, infarct volume and brain edema in brains of MCAO/Rep mice. D-allose inhibited cell apoptosis in the mice model of MCAO/Rep. We observed that D-allose remarkably decreased BBB permeability and prevented the reduction of ZO-1, Occludin and Claudin-5 in mice brains with MCAO/Rep injury. D-allose also repressed the levels of TNF-α, NF-κB, interleukin (IL)-1β and IL-8 in inflammatory responses. The increases of intercellular adhesion molecular-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and CD11b/CD18 were significantly inhibited by D-allose during the MCAO/Rep injury. And D-allose decreased the L-selectin and P-selectin levels after MCAO/Rep. Moreover, D-allose induced up-regulation of peroxisome proliferator-activated receptor γ (PPARγ), and down-regulation of TNF-α and NF-κB after MCAO/Rep, which were abolished by utilization of GW9662. In conclusion, we provided evidences that D-allose may has therapeutic potential against brain IR injury through attenuating BBB disruption and the inflammatory response via PPARγ-dependent regulation of NF-κB.

Huang T ，Gao D ，Hei Y ，Zhang X ，Chen X ，Fei Z ... -  《-》