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 ... -  《-》

GJ-4 ameliorates memory impairment in focal cerebral ischemia/reperfusion of rats via inhibiting JAK2/STAT1-mediated neuroinflammation.

Gardenia jasminoides J. Ellis (Fructus Gardenia) is a traditional Chinese medicine with diverse pharmacological functions, such as anti-inflammation, anti-depression, as well as improvement of cognition and ischemia brain injury. GJ-4 is a natural extract from Gardenia jasminoides J. Ellis (Fructus Gardenia) and has been proved to improve memory impairment in Alzheimer's disease (AD) mouse model in our previous studies. This study aimed to evaluate the therapeutic effects of GJ-4 on vascular dementia (VD) and explore the potential mechanisms. In our experiment, a focal cerebral ischemia and reperfusion rat model was successfully developed by the middle cerebral artery occlusion and reperfusion (MCAO/R). GJ-4 (10 mg/kg, 25 mg/kg, 50 mg/kg) and nimodipine (10 mg/kg) were orally administered to rats once a day for consecutive 12 days. Learning and memory behavioral performance was assayed by step-down test and Morris water maze test. The neurological scoring test was performed to evaluate the neurological function of rats. 2,3,5-Triphenyltetrazolium chloride (TTC) staining and Nissl staining were respectively employed to determine the infarct condition and neuronal injury of the brain. Iba1 immunohistochemistry was used to show the activation of microglia. Moreover, the synaptic damage and inflammatory level were detected by Western blot. GJ-4 could significantly improve memory impairment, cerebral infraction, as well as neurological deficits of VD rats induced by MCAO/R. Further research indicated VD-induced neuronal injury was alleviated by GJ-4. In addition, GJ-4 could protect synapse of VD rats by upregulating synaptophysin (SYP) expression, post synaptic density 95 protein (PSD95) expression, and downregulating N-Methyl-D-Aspartate receptor 1 (NMDAR1) expression. Subsequent investigation of the underlying mechanisms identified that GJ-4 could suppress neuroinflammatory responses, supported by inhibited activation of microglia and reduced expression of inflammatory proteins, which ultimately exerted neuroprotective effects on VD. Further mechanistic study indicated that janus kinase 2 (JAK2)/signal transducer and activator of transcription 1 (STAT1) pathway was inhibited by GJ-4 treatment. These results suggested that GJ-4 might serve as a potential drug to improve VD. In addition, our study indicated that inhibition of neuroinflammation might be a promising target to treat VD.

Liu H ，Zhang Z ，Zang C ，Wang L ，Yang H ，Sheng C ，Shang J ，Zhao Z ，Yuan F ，Yu Y ，Yao X ，Bao X ，Zhang D ... -  《-》

Huanglian Jiedu decoction alleviates ischemia-induced cerebral injury in rats by mitigating NET formation and activiting GABAergic synapses.

Huanglian Jiedu decoction (HLJD) has been used to treat ischemic stroke in clinic. However, the detailed protective mechanisms of HLJD on ischemic stroke have yet to be elucidated. The aim of this study is to elucidate the underlying pharmacological mechanisms of HLJD based on the inhibition of neuroinflammation and the amelioration of nerve cell damage. A middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats and received HLJD treatment. Effects of HLJD on neurological function was assessed based on Bederson's score, postural reflex test and asymmetry score. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining, Hematein and eosin (HE) and Nissl staining were used to observe the pathological changes in brain. Then, transcriptomics was used to screen the differential genes in brain tissue in MCAO/R model rats following HLJD intervention. Subsequently, the effects of HLJD on neutrophil extracellular trap (NET) formation-related neuroinflammation, gamma-aminobutyric acid (GABA)ergic synapse activation, nerve cell damage and proliferation were validated using immunofluorescence, western blot and enzyme-linked immunosorbent assay (ELISA). Our results showed that HLJD intervention reduced the Bederson's score, postural reflex test score and asymmetry score in MCAO/R model rats. Pathological staining indicated that HLJD treatment decreased the cerebral infarction area, mitigated neuronal damage and increased the numbers of Nissl bodies. Transcriptomics suggested that HLJD affected 435 genes in MCAO/R rats. Among them, several genes involving in NET formation and GABAergic synapses pathways were dysregulated. Subsequent experimental validation showed that HLJD reduced the MPO+CitH3+ positive expression area, reduced the protein expression of PAD4, p-P38/P38, p-ERK/ERK and decreased the levels of IL-1β, IL-6 and TNF-α, reversed the increase of Iba1+TLR4+, Iba1+p65+ and Iba1+NLRP3+ positive expression area in brain. Moreover, HLJD increased GABA levels, elevated the protein expression of GABRG1 and GAT3, decreased the TUNEL positive expression area and increased the Ki67 positive expression area in brain. HLJD intervention exerts a multifaceted positive impact on ischemia-induced cerebral injury in MCAO/R rats. This intervention effectively inhibits neuroinflammation by mitigating NET formation, and concurrently improves nerve cell damage and fosters nerve cell proliferation through activating GABAergic synapses.

Cui Y ，Cui M ，Wang L ，Wang N ，Chen Y ，Lv S ，Zhang L ，Chen C ，Yang Y ，Wang F ，Wang L ，Cui H ... -  《-》

The protective role of Tongxinluo on blood-brain barrier after ischemia-reperfusion brain injury.

Tongxinluo (TXL), a renowned traditional Chinese medicine, consists of several different kinds of ingredients and has been widely used to treat myocardial infarction and ischemic stroke. However, the underlying neuroprotective mechanisms are not fully understood. We focus on the effect of TXL on blood-brain barrier (BBB) including edema formation and tight junction (TJ) protein rearrangement, and inflammatory response after transient middle cerebral artery occlusion (tMCAO). We further explore the protective mechanism of TXL on ischemia-induced BBB damage. Adult CD1 male mice (n=168) were randomly divided into TXL pre-treatment group, TXL pre-post treatment group, TXL post-treatment group, control group and sham group. Mice in TXL pre-treatment group were given TXL solution by 1g/kg/day orally for 7 days before tMCAO. Mice in pre-post treatment group were continuously given TXL 7 days before and 14 days after tMCAO. Mice in TXL post-treatment group were given TXL solution immediately after tMCAO. Rotarod test and neurological severity scores were evaluated at 1-14 days following tMCAO. Brains were harvested for examining infarct volume, edema formation, and immunofluorescent staining at 1 and 3 days after tMCAO. Cytokines IL-6, IL-1β and TNF-α mRNA expression, and BBB permeability were further examined by RT-PCR and immunostaining. TXL pre-post treatment improved neurobehavioral outcomes and reduced infarct volume compared to the control (p<0.05). Meanwhile, hemispheric swelling, Evans blue and IgG protein extravasation reduced, while TJ protein expression up-regulated in pre-post treatment group (p<0.05). Further study indicated that infarct volume was smaller and BBB damage was less severe in TXL pre-post treatment group compared to TXL pre-treatment alone. It was noted that fewer myeloperoxidase (MPO) positive cells and less cytokines IL-6, IL-1β and TNF-α expression in pre-post treatment group compared to the control group (p<0.05). TXL pre-treatment and pre-post treatment effectively protected the brain from BBB disruption via alleviating inflammatory response. Moreover, pre-post treatment has better outcomes, suggesting that continuous administration of TXL before and throughout ischemia period is necessary because of multiple functions of TXL.

Liu Y ，Tang GH ，Sun YH ，Lin XJ ，Wei C ，Yang GY ，Liu JR ... -  《-》

JR14a: A novel antagonist of C3aR attenuates neuroinflammation in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI), a prevalent stroke-related complication, can lead to severe brain damage. Inflammation is a crucial factor in CIRI pathogenesis, and the complement component 3a receptor (C3aR) could be a key mediator in the post-CIRI inflammatory cascade. In this study, the role of C3aR in CIRI was investigated utilizing a middle cerebral artery occlusion (MCAO) model in C3aR knockout (KO) mice. Magnetic resonance imaging (MRI) and neurofunctional assessments revealed that C3aR KO mice exhibited significantly diminished cerebral infarction and improved neurological impairments. Consequently, the focus shifted to searching for a small molecule antagonist of C3aR. JR14a, a new potent thiophene antagonist of C3aR, was injected intraperitoneally into mice 1-h post-MCAO model implementation. The mass spectrometry (MS) results indicated the ability of JR14a to penetrate the blood-brain barrier. Subsequent TTC staining and neurofunctional assessments revealed the efficacy of JR14a in reducing cerebral infarct volume and neurological impairment following MCAO. In addition, immunofluorescence (IF) and immunohistochemistry (IHC) demonstrated attenuated microglial activation, neutrophil infiltration, and blood-brain barrier disruption by JR14a in the MCAO model. Furthermore, enzyme-linked immunosorbent assay (ELISA) and Western blotting supported the role of JR14a in downregulating the expression levels of C3aR, tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), as well as the phosphorylation of p65. In conclusion, the findings suggested that C3aR could be a potential therapeutic target for CIRI, and JR14a emerged as a promising treatment candidate.

Tang J ，Maihemuti N ，Fang Y ，Tan J ，Jia M ，Mu Q ，Huang K ，Gan H ，Zhao J ... -  《-》