Buyang Huanwu decoction promotes angiogenesis after cerebral ischemia through modulating caveolin-1-mediated exosome MALAT1/YAP1/HIF-1α axis.

BACKGROUND: Angiogenesis is an effective method for promoting neurological function recovery after cerebral ischemia (CI). Buyang Huanwu decoction (BHD) is a traditional Chinese medicinal recipe that is frequently employed for CI treatment. Previous investigations have validated that it promotes angiogenesis following CI. Nevertheless, the precise mechanism by which it does this has yet to be completely understood. OBJECTIVE: This study aims to examine the underlying mechanism through which BHD facilitates angiogenesis following CI by regulating the exosomal MALAT1/YAP1/HIF-1α signaling axis, specifically via the involvement of caveolin-1 (Cav1), an endocytosis-associated protein. METHODS: A CI model was created using middle cerebral artery occlusion (MCAO). Following the administration of multiple doses of BHD, various parameters, including the neurobehavioral score, pathological damage, and angiogenesis, were assessed in each group of mice to identify the optimal dosage of BHD for treating CI. The molecular processes underlying the angiogenic implications of BHD following CI were investigated exhaustively by employing single-cell sequencing. Finally, the involvement of Cav1 was confirmed in Cav1 knockout mice and Cav1-silenced stably transfected strains to validate the mechanism by which BHD increases angiogenesis following CI. RESULTS: BHD could promote angiogenesis after CI. Single-cell sequencing results suggested that its potential mechanism of action might be connected with Cav1 and the exosomal MALAT1/YAP1/HIF-1α signaling axis. BHD could promote angiogenesis after CI by regulating the exosomal MALAT1/YAP1/HIF-1α axis through Cav1, as validated in vivo and in vitro experiments. Accordingly, Cav1 may be a key target of BHD in promoting angiogenesis after CI. CONCLUSION: This investigation represents the initial attempt to comprehensively ascertain the underlying mechanism of action of BHD in treating CI using single-cell sequencing, gene-knockout mice, and stable transfected cell lines, potentially associated with the modulation of the exosomal MALAT1/YAP1/HIF-1α axis by Cav1. Our findings offer novel empirical evidence for unraveling the regulatory pathways through which Cav1 participates in angiogenesis following CI and shed light on the potential mechanisms of BHD.

N6-methyladenosine-modified lncRNA and mRNA modification profiles in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) is common in ischemic stroke and seriously affects the prognosis of patients. At present, N6-methyladenosine (m6A) modification of lncRNAs and mRNAs has been reported in other diseases, such as cancer, but its role in CIRI has not been clarified. In this study, we aimed to investigate the m6A lncRNA and m6A mRNA modification profiles in CIRI. First, we detected the total level of m6A and the changes in related m6A methyltransferases and demethylases in the brain tissue of rats with CIRI and then identified differentially modified lncRNAs and mRNAs in CIRI by lncRNA and mRNA epigenetic transcriptomic microarray. In addition, bioinformatics analysis was used to predict the underlying functions and related pathways of related lncRNAs and mRNAs. We found that the total m6A methylation level was significantly increased, and the expression of fat mass and obesity-associated protein (FTO) was downregulated after CIRI. In addition, a large number of m6A-modified lncRNAs and mRNAs appeared after CIRI, and these genes were mainly enriched for the Toll-like receptor signaling pathway, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway. Our findings provide the basis and insights for further studies on m6A modification in CIRI.

[Effect of Naoshuming decoction on expression of NF-κB, TNF-α, and IL-1ß in focal cerebral ischemia rats].

OBJECTIVE: To explore the effect of Naoshuming decoction on cerebral ischemic rats.
 Methods: The model of cerebral ischemia in rats was established via middle cerebral artery occlusion (MCAO). The MCAO model rats were randomly divided into a model group (n=36), a Naoshuming decoction at high dose group (n=36), a Naoshuming decoction at middle dose group (n=36) and a Naoshuming decoction at low dose group (n=36). In addition, a normal group (n=12) and a sham operation group (n=12) were included. Rats in each group were killed on the 3rd, 7th, and 14th day to detect relevant indicators. The Ayelet Levy 14 method was used to score the neurological function. Immunohistochemical method was used to detect the protein expression of nuclear factor kappa-B (NF-κB)/p50, NF-κB/p65, tumor necrosis factor-α (TNF-α), and IL-1ß. The quantitative real-time PCR were used to detect the mRNA expression of NF-κB, TNF-α and IL-1ß. 
 Results: Compared with the sham group, at each time point, the inflammation indexes in the model group and different dose of Naoshuming decoction groups were significantly enhanced, and all of them showed neurological dysfunction. But the inflammatory indexes and neurological function scores would were gradually improved with the pass of time. Compared with the model group, the neurological dysfunction, the protein levels of NF-κB/p50, NF-κB/p65, TNF-α and IL-1ß, and the mRNA of NF-κB, TNF-α and IL-1ß in the high, middle and low dose of Naoshuming decoction groups were reduced at 3, 7 and 14 d, with statistical difference (all P<0.05 or P<0.01). 
 Conclusion: Naoshuming decoction can alleviate the cerebral ischemic injury in rats.

Flagellar Hooks and Hook Protein FlgE Participate in Host Microbe Interactions at Immunological Level.

Host-microbe interactions determine the outcome of host responses to commensal and pathogenic microbes. Previously, two epithelial cell-binding peptides were found to be homologues of two sites (B, aa168-174; F, aa303-309) in the flagellar hook protein FlgE of Pseudomonas aeruginosa. Tertiary modeling predicted these sites at the interface of neighboring FlgE monomers in the fully formed hook. Recombinant FlgE protein stimulated proinflammatory cytokine production in a human cell line and in murine lung organoid culture as detected with real-time RT-PCR and ELISA assays. When administered to mice, FlgE induced lung inflammation and enhanced the Th2-biased humoral response to ovalbumin. A pull-down assay performed with FlgE-saturated resin identified caveolin-1 as an FlgE-binding protein, and caveolin-1 deficiency impaired FlgE-induced inflammation and downstream Erk1/2 pathway activation in lung organoids. Intact flagellar hooks from bacteria were also proinflammatory. Mutations to sites B and F impaired bacteria motility and proinflammatory potency of FlgE without altering adjuvanticity of FlgE. These findings suggest that the flagellar hook and FlgE are novel players in host-bacterial interactions at immunological level. Further studies along this direction would provide new opportunities for understanding and management of diseases related with bacterial infection.

Buyang Huanwu Decoction Ameliorates Poststroke Depression via Promoting Neurotrophic Pathway Mediated Neuroprotection and Neurogenesis.

Objective. The aim of the present research is to investigate the therapeutic effect of Buyang Huanwu Decoction (BHD) in poststroke depression (PSD) animal model and illustrate its underlying mechanism via promoting neurotrophic pathway mediated neuroprotection and neurogenesis. Methods. To induce PSD rat model, isolation housed rats that received middle cerebral artery occlusion (MCAO) surgery successively suffered from chronic mild stress (CMS) treatment for consecutive twenty-one days. Meanwhile, rats were correspondingly given vehicle, BHD, and fluoxetine. Then, neurologic function was scored and depressive-like behaviors were assessed by sucrose preference test, locomotor activity, novelty-suppressed feeding test, and forced swim test. Thereafter, the neuroprotection and neurogenesis related molecular markers and signaling were detected. Results. We firstly observed a significant neurological function recovery and antidepressants effect of BHD after MCAO together with CMS treatment. Our study also found that treatment with BHD and fluoxetine can significantly rescue neurons from apoptosis and promote neurogenesis in the CA3 and DG regions in the hippocampus. Notably, BHD and fluoxetine treatment can activate BDNF/ERK/CREB signaling. Conclusion. The results suggest that BHD is a promising candidate for treating PSD. Its curative effects can be attributed to neurotrophic pathway mediated neuroprotection and neurogenesis.

Buyang Huanwu Decoction () reduces infarct volume and enhances estradiol and estradiol receptor concentration in ovariectomized rats after middle cerebral artery occlusion.

OBJECTIVE: To investigate the effect of Buyang Huanwu Decoction (, BYHWD) on estradiol (E2) and estradiol receptor (ER) in serum and brain in ovariectomized rats after middle cerebral artery occlusion (MCAO). METHODS: Adult female rats were ovariectomized and focal cerebral ischemic was induced by MCAO. Rats were randomly divided into normal, ovariectomy (OVX), MCAO, OVX+MCAO, OVX+MCAO+E2, and OVX+MCAO+BYHWD group. Rats were administered BYHWD 5 g/kg daily, estradiol valerate 500 µg/kg per day or distilled water for 7 consecutive days. Neuronal function and infarct volume were measured on day 7 after artery occlusion, and E2 and ER concentration in serum and brain were checked by enzyme-linked immunosorbent assay. RESULTS: BYHWD significantly improved the neurological behavior, reduced the infarction volume, increased E2 concentration in serum and brain, and increased ER concentration in the brain in ovariectomized rats after MCAO. CONCLUSION: The neuroprotective effects of BYHWD are associated with estrogen and its receptor.

[Effect of ultra-micronized Buyang Huanwu decoction on neurological function, quality of life, and serum vascular endothelial growth factor in patients convalescent from cerebral infarction].

OBJECTIVE: To study effect of Buyang Huanwu decoction (BYHWD) on neurological function, quality of life, and serum vascular endothelial growth factor (VEGF) in patients convalescent from cerebral infarction, and to evaluate the effect of ultra-micronized BYHWD. METHODS: Two hundred and fifty-one patients met the inclusion criteria were randomly assigned to traditional BYHWD (TB) group (n=83), ultra-micronized BYHWD (UB) group (n=85) and the control group (n=83) according to time of entrance into the study with 1:1:1. All patients received rehabilitation training, but for patients in the TB and UB groups, traditional BYHWD (15 g, twice a day) or ultra-micronized BYHWD (5 g, twice a day) was given respectively, for a course of 12 weeks. Clinical curative effect and curative effect of syndrome according to traditional Chinese medicine (TCM) were evaluated. Nerve function and quality of life in patients were evaluated, serum VEGF was determined before and after treatment. The level of VEGF in 23 healthy volunteers was also determined to serve as normal control. RESULTS: The total effective rate was 83.5%, 85.5% and 77.1% in UB group, TB group and the control group, respectively, and the total symptomatic effective rate in TCM was 87.0%, 89.2% and 77.1%, respectively. Compared with the control group, there was significant difference in UB or TB group (all P<0.05), but there was no significant difference between UB and TB groups (both P >0.05). Serum VEGF levels (ng/L) were significantly lower before treatment in control group, TB group and UB group than those in normal control group (79.87±2.81, 80.19±3.23, 80.23±3.18 vs. 68.13±3.39, all P<0.05). Neurologic deficit score (NDS), quality of life and serum VEGF were improved after treatment in three groups, but they were better in UB or TB group than the control group [NDS: 11.95±5.03, 12.68±4.67 vs. 15.23±5.12, quality of life score: 64.71±6.73, 63.56±6.53 vs. 59.09±6.81, serum VEGF (ng/L): 76.38±3.02, 76.84±3.18 vs. 70.26±3.15 , all P<0.05], but there was no significant difference between UB and TB groups (all P >0.05). CONCLUSION: BYHWD can improve neurological function and quality of life, and increase serum VEGF in patients convalescent from cerebral infarction, and ultra-micronized BYHWD, the dosage can be decreased.

[Effect of Buyang Huanwu decoction on interleukin-1ß and tumor necrosis factor-α expression in rats after cerebral infarction].

OBJECTIVE: To explore the effect of Buyang Huanwu decoction (BYHWD) on pro-inflammatory cytokines in rats after focal cerebral infarction. METHODS: Adult Sprague Dawley (SD) rats were randomly divided into following groups: normal control, sham, model, BYHWD. The rats in latter three groups were subdivided into subgroups of 1, 3, and 7 days after medication, with 5 rats in each group. The right side focal cerebral infarction model was reproduced by middle cerebral artery occlusion (MCAO). The rats in BYHWD group were gavaged with BYHWD of 10 ml/kg (14.2 g/kg, once a day) 2 hours after operation. Animals were sacrificed at corresponding time points. The protein and mRNA expression of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: There were low levels expression of IL-1ß and TNF-α protein and mRNA in normal control group and the sham group. After cerebral infarction, the protein and mRNA expression of IL-1ß and TNF-α began to increase in rats 1 day after the insult, and the protein and mRNA expression of IL-1ß reached the peak on 3rd day, and then lowered, and the protein and mRNA expression of TNF-α reached the peak on 7th day. Compared with model group on 1st, 3rd and 7th day, the protein expression of IL-1ß (ng/L: 90.290±8.693 vs. 102.556±13.934 on 1st day, 129.632±11.050 vs. 150.117±8.552 on 3rd day, 66.185±9.020 vs. 91.362±9.901 on 7th day) and TNF-α (ng/L: 210.341±19.247 vs. 236.887±20.137 on 1st day, 267.503±21.006 vs. 322.659±15.068 on 3rd day, 299.637±17.717 vs. 386.678±16.297 on 7th day), and mRNA expression of IL-1ß (1 day: 0.54±0.09 vs. 0.64±0.11, 3 days: 0.80±0.06 vs. 0.89±0.07, 7 days : 0.70±0.09 vs. 0.78±0.08) and TNF-α (1 day: 0.64±0.09 vs. 0.73±0.11, 3 days: 0.74±0.13 vs. 0.85±0.07 , 7 days : 0.82±0.07 vs. 0.93±0.08], were all decreased obviously in BYHWD group ( P<0.05 or P<0.01). CONCLUSION: BYHWD could reduce the protein and mRNA expressions of IL-1ß and TNF-α in levels after cerebral infarction. The result shows that it protects brain by modulating expression of pro-inflammatory mediators.

Buyang Huanwu Decoction can improve recovery of neurological function, reduce infarction volume, stimulate neural proliferation and modulate VEGF and Flk1 expressions in transient focal cerebral ischaemic rat brains.

Buyang Huanwu Decoction is a classic formula for treating stroke-induced disability in traditional Chinese medicine (TCM). To explore its pharmacological basis, we investigated the effects of the whole formula and its herbal components on the neurological behavior performance and infarction volume in focal cerebral ischaemia rats. The neurological deficit scores and infarction volume were measured at days 3, 7 and 14 after 30 min of occlusion of middle cerebral artery. The results showed that Buyang Huanwu Decoction and its herbal components significantly improved the neurological behavior performances and reduced the infarction volume in the ischaemic brains. To elucidate the potential therapeutic mechanisms, we investigated the proliferation of progenitors by detecting the immunohistochemical staining of thymidine analog 5-bromo-2'-deoxyuridine (BrdU) and found that the formula stimulated the proliferation of the progenitors at hippocampus and subventricular zone (SVZ) in the ischaemic brains. As vascular endothelial growth factor (VEGF) and its receptor fetal liver kinase (Flk1) are important neurotrophic, neuroprotective and neuroproliferative factors, we studied the expressions of VEGF and Flk1 in the hippocampus, SVZ and cortex in the ischaemic brains and found that the formula led to increase the numbers of VEGF-positive and Flk1-positive cells in the SVZ and cortex in the ischaemic brains. The results indicate that the therapeutic effects of Buyang Huanwu Decoction for recovery of neurological deficits are associated with the stimulation of the proliferation of progenitors and the enhancement of the expressions of VEGF and Flk in ischaemic brains.

[Effects of nao-yi-an on tumor necrosis factor alpha and insulin resistance of acute intracerebral hemorrhagic patients].

OBJECTIVE: To investigate the effects of nao-yi-an on tumor necrosis factor alpha (TNF-alpha) and insulin resistance of acute intracerebral hemorrhagic patients. METHODS: The patients were randomly divided into two groups: the medicine control group and the nao-yi-an treatment group. TNF-alpha and insulin were checked by radioimmunoassay before and after the treatment. Fifteen healthy people were made as the negative control group. RESULTS: TNF-alpha of both treatment groups was higher while insulin sensitivity index(ISI) was remarkably lower compared with the health group (P < 0.01). After the treatment ISI increased and TNF-alpha was remarkably lower in the treatment group (P < 0.01). Differences of TNF-alpha and ISI between the treatment group and medicine control group were significant (P < 0.05 or P < 0.01). CONCLUSION: nao-yi-an can decrease TNF-alpha and increase ISI, which is a mechanism of reducing cerebral ischemic-reperfusion injury.