Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair.

OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 ß in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 ß (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS: DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.

Exploring the synergic mechanism of Ligusticum striatum DC. and borneol in attenuating BMECs injury and maintaining tight junctions against cerebral ischaemia based on the HIF-1α/VEGF signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum striatum DC., also known as Ligusticum chuanxiong Hort. (LCH), is widely used in China for its excellent effect in ischaemic stroke (IS) patients, and borneol (BO) has been confirmed to maintain the blood‒brain barrier (BBB) after stroke. They are often used as a combination in the prescriptions of IS patients. Although the advantage of their combined treatment in improving brain ischaemia has been verified, their synergistic mechanism on BBB maintenance is still unclear. AIM OF THE STUDY: This study was designed to evaluate the synergistic effect of maintaining the BBB between LCH and BO against IS and to further explore the potential mechanism. MATERIALS AND METHODS: After primary mouse brain microvascular endothelial cells (BMECs) were extracted and identified, the duration of oxygen-glucose deprivation (OGD) and the doses of LCH and BO were optimized. Then, the cells were divided into five groups: control, model, LCH, BO, and LCH + BO. Cell viability, injury degree, proliferation and migration were detected by CCK-8, LDH, EdU and wound-healing assays, respectively. Hoechst 33342 staining was adopted to detect the apoptosis rate, and western blotting was employed to observe the expressions of Bax, Bcl-2, caspase-3 and cleaved caspase-3. The TEER value and NaF permeability were measured to assess tight junction (TJ) function, while ZO-1, occludin and claudin-5 were also probed by western blotting. Moreover, the HIF-1α/VEGF pathway was observed to explore the underlying mechanism of BBB maintenance. In vivo, global cerebral ischaemia/reperfusion (GCIR) surgery was performed to establish an IS model. After treatment with LCH (200 mg/kg) and/or BO (160 mg/kg), histopathological structure and BMECs repair were observed by HE staining and immunohistochemistry of vWF. Meanwhile, TJ-associated proteins in vivo were also detected by western blotting. RESULTS: Basically, LCH and BO had different emphases. LCH significantly attenuated the vacuolar structure, nuclear pyknosis and neuronal loss of GCIR mice, while BO focused on promoting BMECs proliferation and angiogenesis and inhibiting the degradation of TJ-associated proteins in vivo after IS. Interestingly, their combination further enhanced these effects. OGD injury markedly reduced the viability, proliferation and migration of primary BMECs; decreased the ratio of Bcl-2/Bax, TEER value, and the expressions of ZO-1, occludin and claudin-5; induced LDH release and apoptosis; and increased the cleaved caspase-3/caspase-3 ratio and NaF permeability. Meanwhile, BO might be the main contributor to the combinative treatment in ameliorating OGD-induced damage of BMECs and degradation of TJ-related proteins, and the potential mechanism might be involved in upregulating the HIF-1α/VEGF signalling pathway. Although LCH showed no obvious improvement, it could enhance the therapeutic effect of BO. Interestingly, their combination even produced some new improvements, including the reduction of cleaved caspase-3 and increase in TEER value, none of which were exhibited in their monotherapies. CONCLUSIONS: LCH and BO exhibited complementary therapeutic features in alleviating cerebral ischaemic injury by inhibiting BMECs apoptosis, maintaining the BBB and attenuating the loss of neurons. LCH preferred to protect ischaemic neurons, while BO played a key role in protecting BMECs, maintaining the BBB and TJs by activating the HIF-1α/VEGF signalling pathway.

Essential oil of Ligusticum chuanxiong Hort. Regulated P-gp protein and tight junction protein to change pharmacokinetic parameters of temozolomide in blood, brain and tumor.

ETHNOPHARMACOLOGICAL RELEVANCE: The existence of the blood-brain barrier/blood tumor barrier (BBB/BTB) severely restricts the effectiveness of anti-tumor drugs, thus glioma is still an incurable disease with a high fatality rate. Chuanxiong (Ligusticum chuanxiong Hort., Umbelliferae) was used as a messenger drug to increase the distribution of drugs in brain tissue, and its application in Chinese herbal formula for treating glioma was also the highest. AIM OF THE STUDY: Our previous researches showed that essential oil (EO) of chuanxiong could promote temozolomide (TMZ) entry into glioma cells in vitro and enhance TMZ-induced anticancer efficiency in vivo, and therefore, the aim of this study was to investigate whether EO could increase the concentration accumulation of TMZ in brain or tumor of C6 glioma rats and the related mechanisms. MATERIALS AND METHODS: The pharmacokinetics were conducted in C6 glioma rats by administering either TMZ alone or combined with EO through oral routes. TMZ concentration in blood, brain and tumor was detected using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and then pharmacokinetic parameters were calculated. The changed expressions of P-gp protein, tight junction occludin, claudin-5 and zonula occludens-1 (ZO-1) in brain of glioma rats were studied by Western blot to clarify the mechanism. Finally, the chemical composition of EO was analyzed by gas chromatography-massspectrometry (GC-MS). RESULTS: The results showed that EO significantly affected the pharmacokinetic parameters such as Tmax, Cmax and CL (p < 0.01), but did not significantly change the AUC(0→∞) of TMZ in blood (p > 0.05). However, EO markedly improved the AUC(0→∞)of TMZ in brain and tumor (p < 0.01). The calculate drug targeting index was greater than 1, indicating that EO could promote the distribution of TMZ to the brain and tumor. Western blot analysis showed that EO significantly inhibited the expression of P-gp, tight junction protein claudin-5, occludin and ZO-1. And meanwhile, the expressions of P-gp, claudin-5 and occludin also markedly down-regulated in EO-TMZ co-administration treatment. GC-MS analysis of the TIC component of EO was (E)-Ligustilide (36.93%), Terpinolene (7.245%), gamma-terpinene (7.225%) etc. CONCLUSION: EO could promote the distribution of TMZ in the brain and tumor of C6 glioma rats, which may attribute to down-regulate the expression of P-gp, claudin-5 and occludin.

[Therapeutic effect of Shengjiang Powder on experimental autoimmune encephalomyelitis mice through IL-6/JAK2/STAT3 signaling pathway:based on HPLC].

A high performance liquid chromatography(HPLC) method was established to analyze the components in Shengjiang Powder(SJP) such as emodin and curcumin and explore its therapeutic effect on experimental autoimmune encephalomyelitis(EAE) mice. To be specific, HPLC was performed to determine the content of compounds in SJP such as emodin and curcumin. A total of 72 female SPF C57 BL/6 mice were randomized into control group(equivalent volume of ultrapure water, ig), model group(equivalent volume of ultrapure water, ig), low-, medium-, and high-dose SJP groups(SJP, ig), and positive control group(prednisone acetate, ig), 12 each group. EAE was induced in mice except the control group. Administration began from the first day after immunization. The general conditions, symptom score, and body weight of the mice were recorded. On the 21 st day, mouse brain tissues were separrated. Then hematoxylin-eosin(HE) staining and Luxol Fast Blue(LFB) staining were used to detect the pathological changes of brain tissues. Immunohistochemistry(IHC) was employed to determine the myelin basic protein(MBP) level, and Western blot the expression of occludin and claudin-5, as well as the levels of interleukin-6(IL-6) and proteins in the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3) pathway and their phosphorylation levels. The mRNA expression of IL-6, JAK2, and STAT3 was detected by real-time quantitative polymerase chain reaction(qPCR). Finally, molecular docking of six main active components in SJP, including emodin and curcumin, with IL-6, JAK2 and STAT3 was performed, and the binding affinity was evaluated. The results showed that the established HPLC method demonstrated high precision, reproducibility, stability, and high recovery of samples. Compared with the model group, SJP reduced the clinical symptom score and alleviate the inflammatory infiltration of brain white matter and demyelination of EAE mice. At the same time, SJP increased the expression of occludin and claudin-5, down-regulated the mRNA expression of IL-6, JAK2, and STAT3, as well as the levels of IL-6/JAK/STAT3 proteins and the phosphorylation levels, with significant difference. Molecular docking suggested that the six active components in SJP had high binding energy with IL-6, JAK2, and STAT3 proteins. The established HPLC method is simple, accurate, and highly sensitive, which can simultaneously determine the content of emodin and curcumin in SJP. SJP may alleviate the clinical symptoms of EAE by inhibiting IL-6/JAK2/STAT3 signaling pathway, protecting the blood-brain barrier, and relieving the inflammatory response and demyelinization of brain tissue.

Wenyang Huazhuo Tongluo formula alleviates pulmonary vascular injury and downregulates HIF-1α in bleomycin-induced systemic sclerosis mouse model.

BACKGROUND: Vascular damage, autoimmune abnormalities, and fibrosis are the three pathological features of systemic sclerosis (SSc).However, pulmonary vascular damage is the main factor affecting the progression and prognosis of SSc. The main purpose of this study was to explore the molecular mechanism of Wenyang Huazhuo Tongluo Formula in alleviating pulmonary vascular injury in bleomycin-induced SSc mouse model. METHODS: Masson staining and H&E staining were used to analyze the degree of pulmonary vascular fibrosis and the infiltration of leukocyte cells in lung tissue ofbleomycin-induced SSc mouse models treated with saline (BLM group), Wenyang Huazhuo Tongluo Formula (WYHZTL group) and HIF-1α inhibitor KC7F2 (KC7F2 group). Blood vessel exudation was determined by analyzing the cell number and albumin concentration in bronchoalveolar lavage fluid using a cell counter and ELISA assay, respectively. The degree of vascular injury was assessed by measuring the expression levels of vWF, E-selectin, ICAM-1, VCAM-1, VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells using ELISA and immunofluorescence staining. Finally, the effect of Wenyang Huazhuo Tongluo Formula on the expression of HIF-1α was detected using immunofluorescence staining. RESULTS: Wenyang Huazhuo Tongluo Formula and KC7F2 significantly inhibited bleomycin-induced pulmonary vascular fibrosis and the level of perivascular inflammatory cell infiltration. The number of cells and the concentration of albumin were significantly reduced in the bronchoalveolar lavage fluid of the WYHZTL group and KC7F2 group compared with the BLM group. In addition, treatment with Wenyang Huazhuo Tongluo Formula and KC7F2 significantly downregulated the expression levels of vWF, E-selectin, ICAM-1, VCAM-1 and HIF-1α, but upregulated the expression of VE-cadherin and claudin-5 in serum and pulmonary vascular endothelial cells, compared with treatment with saline. CONCLUSIONS: This study reveals that Wenyang Huazhuo Tongluo Formula plays a new role in the treatment of SSc by alleviating pulmonary vascular damage. Furthermore, we found that Wenyang Huazhuo Tongluo Formula alleviates pulmonary vascular injury and inhibits HIF-1α expression.

Effects of cannabinoid (CBD) on blood brain barrier permeability after brain injury in rats.

Cannabidiol is a natural herbal medicine known to protect the brain from traumatic brain injury (TBI). Here, a TBI rat model was established, with cannabidiol administered intraperitoneally at doses of 5, 10, or 20 mg/kg, 30 min before surgery and 6 h after surgery until sacrifice. Brain water content, body weight, and modified neurological severity scores were determined, and enzyme-linked immunosorbent assay, immunofluorescence staining, hematoxylin and eosin staining, Nissl staining, Evans-blue dye extravasation, and western blotting were performed. Results showed that cannabidiol decreased the number of aquaporin-4-positive and glial fibrillary acidic protein-positive cells. Cannabidiol also significantly reduced the protein levels of proinflammatory cytokines (TNF-α and IL-1ß) and significantly increased the expression of tight junction proteins (claudin-5 and occludin). Moreover, cannabidiol administration significantly mitigated water content in the brain after TBI and blood-brain barrier disruption and ameliorated the neurological deficit score after TBI. Cannabidiol administration improved the integrity and permeability of the blood-brain barrier and reduced edema in the brain after TBI.

Comparative assessment of in vitro BBB tight junction integrity following exposure to cigarette smoke and e-cigarette vapor: a quantitative evaluation of the protective effects of metformin using small-molecular-weight paracellular markers.

BACKGROUND: The blood-brain barrier (BBB) plays a critical role in protecting the central nervous system (CNS) from blood-borne agents and potentially harmful xenobiotics. Our group's previous data has shown that tobacco smoke (TS) and electronic cigarettes (EC) affect the BBB integrity, increase stroke incidence, and are considered a risk factor for multiple CNS disorders. Metformin was also found to abrogate the adverse effects of TS and EC. METHODS: We used sucrose and mannitol as paracellular markers to quantitatively assess TS and EC's impact on the BBB in-vitro. Specifically, we used a quantitative platform to determine the harmful effects of smoking on the BBB and study the protective effect of metformin. Using a transwell system and iPSCs-derived BMECs, we assessed TS and EC's effect on sucrose and mannitol permeability with and without metformin pre-treatment at different time points. Concurrently, using immunofluorescence (IF) and Western blot (WB) techniques, we evaluated the expression and distribution of tight junction proteins, including ZO-1, occludin, and claudin-5. RESULTS: Our data showed that TS and EC negatively affect sucrose and mannitol permeability starting after 6 h and up to 24 h. The loss of barrier integrity was associated with a reduction of TEER values. While the overall expression level of ZO-1 and occludin was not significantly downregulated, the distribution of ZO-1 was altered, and discontinuation patterns were evident through IF imaging. In contrast to occludin, claudin-5 expression was significantly decreased by TS and EC, as demonstrated by WB and IF data. CONCLUSION: In agreement with previous studies, our data showed the metformin could counteract the negative impact of TS and EC on BBB integrity, thus suggesting the possibility of repurposing this drug to afford cerebrovascular protection.

Daidzein ameliorates experimental acute reflux esophagitis in rats via regulation of cytokines.

Context: Daidzein is a secondary metabolite derived from plants, has a flavonoid structure and is known for its protective activity in gastrointestinal disorders. Objective: The current work determines the preventive effect of daidzein against injury in the esophagus mucosa induced by esophageal reflux (RE) in an animal model. Methods: Adult male Wistar rats were classified into six groups: normal control, ER + different doses of daidzein and ER + omeprazole. RE was induced in all animals except controls and supplemented with daidzein and standard drugs orally for 6 hours. Serum and tissue were used for further biochemical parameters. Results: Daidzein as a flavonoid has antioxidant properties and shows in vitro antioxidant activity. The outcomes also reveal an elevation in lipid peroxidation and a decline in the levels of sulphhydryl groups and glutathione, along with the depletion in the activities of enzymatic antioxidants in the oxidative stress state. In a dose-dependent manner daidzein and omeprazole amended all macroscopic and biochemical variations and protected against the raised level of hydrogen peroxide (H2O2), calcium and free iron levels in esophageal tissue induced during RE. It also improved the expression and level of proinflammatory cytokines. Conclusion: The finding reports that daidzein has a potential to show a shielding effect against esophagus damage induced by RE in rats, at least in part via alteration of inflammatory cytokines.

Alanyl-Glutamine Restores Tight Junction Organization after Disruption by a Conventional Peritoneal Dialysis Fluid.

Understanding and targeting the molecular basis of peritoneal solute and protein transport is essential to improve peritoneal dialysis (PD) efficacy and patient outcome. Supplementation of PD fluids (PDF) with alanyl-glutamine (AlaGln) increased small solute transport and reduced peritoneal protein loss in a recent clinical trial. Transepithelial resistance and 10 kDa and 70 kDa dextran transport were measured in primary human endothelial cells (HUVEC) exposed to conventional acidic, glucose degradation products (GDP) containing PDF (CPDF) and to low GDP containing PDF (LPDF) with and without AlaGln. Zonula occludens-1 (ZO-1) and claudin-5 were quantified by Western blot and immunofluorescence and in mice exposed to saline and CPDF for 7 weeks by digital imaging analyses. Spatial clustering of ZO-1 molecules was assessed by single molecule localization microscopy. AlaGln increased transepithelial resistance, and in CPDF exposed HUVEC decreased dextran transport rates and preserved claudin-5 and ZO-1 abundance. Endothelial clustering of membrane bound ZO-1 was higher in CPDF supplemented with AlaGln. In mice, arteriolar endothelial claudin-5 was reduced in CPDF, but restored with AlaGln, while mesothelial claudin-5 abundance was unchanged. AlaGln supplementation seals the peritoneal endothelial barrier, and when supplemented to conventional PD fluid increases claudin-5 and ZO-1 abundance and clustering of ZO-1 in the endothelial cell membrane.

Inducers of the endothelial cell barrier identified through chemogenomic screening in genome-edited hPSC-endothelial cells.

The blood-retina barrier and blood-brain barrier (BRB/BBB) are selective and semipermeable and are critical for supporting and protecting central nervous system (CNS)-resident cells. Endothelial cells (ECs) within the BRB/BBB are tightly coupled, express high levels of Claudin-5 (CLDN5), a junctional protein that stabilizes ECs, and are important for proper neuronal function. To identify novel CLDN5 regulators (and ultimately EC stabilizers), we generated a CLDN5-P2A-GFP stable cell line from human pluripotent stem cells (hPSCs), directed their differentiation to ECs (CLDN5-GFP hPSC-ECs), and performed flow cytometry-based chemogenomic library screening to measure GFP expression as a surrogate reporter of barrier integrity. Using this approach, we identified 62 unique compounds that activated CLDN5-GFP. Among them were TGF-ß pathway inhibitors, including RepSox. When applied to hPSC-ECs, primary brain ECs, and retinal ECs, RepSox strongly elevated barrier resistance (transendothelial electrical resistance), reduced paracellular permeability (fluorescein isothiocyanate-dextran), and prevented vascular endothelial growth factor A (VEGFA)-induced barrier breakdown in vitro. RepSox also altered vascular patterning in the mouse retina during development when delivered exogenously. To determine the mechanism of action of RepSox, we performed kinome-, transcriptome-, and proteome-profiling and discovered that RepSox inhibited TGF-ß, VEGFA, and inflammatory gene networks. In addition, RepSox not only activated vascular-stabilizing and barrier-establishing Notch and Wnt pathways, but also induced expression of important tight junctions and transporters. Taken together, our data suggest that inhibiting multiple pathways by selected individual small molecules, such as RepSox, may be an effective strategy for the development of better BRB/BBB models and novel EC barrier-inducing therapeutics.

Components identification and nutritional value exploration of tea (Camellia sinensis L.) flower extract: Evidence for functional food.

Camellia sinensis L., its fresh leaves and buds are used to make tea, is an important industrial crop with a long history. However, less attention has been paid to tea flowers. Indeed, tea flower extract (TFE) is a rich source of functional molecules, but its nutritional value remains unclear. This study, from the perspective of "whole food", aimed to investigate the composition of TFE and further explore its possible health-promoting effects on cyclophosphamide-induced mice. It was found that TFE was mainly composed of carbohydrates (34.02 ± 1.42%), phenolic compounds (11.57 ± 0.14%), crude proteins (27.72 ± 3.07%) and saponins (2.81 ± 0.00%). Supplementation of TFE at 200 mg/kg·BW/d regulated intestinal homeostasis by improving the intestinal barrier, alleviating dysbacteriosis (reverse 44 of 68 disordered genera), stimulated immunoreactions with significant enhancement of serum TNF-α, IFN-γ, IL-1ß, IL-2 and IL-6. Furthermore, TFE could improve the liver function through decreasing the hepatic malondialdehyde and aminotransferase levels and increasing the levels of catalase, myeloperoxidase, superoxide dismutase and reduced glutathione. Notably, the ameliorating effects of TFE on cyclophosphamide-induced immunosuppression and the hepatic injury were associated with its modulation of gut microbiota. The results provide the evidence for the application of tea flower as potential functional food.

Shengui Sansheng Pulvis maintains blood-brain barrier integrity by vasoactive intestinal peptide after ischemic stroke.

Background Shengui Sansheng Pulvis (SSP) has about 300 years history used for stroke treatment, and evidences suggest it has beneficial effects on neuro-angiogenesis and cerebral energy metabolic amelioration post-stroke. However, its protective action and mechanisms on blood-brain barrier (BBB) is still unknown. Purpose Based on multiple neuroprotective properties of vasoactive intestinal peptide (VIP) in neurological disorders, we investigate if SSP maintaining BBB integrity is associated with VIP pathway in rat permanent middle cerebral artery occlusion (MCAo) model. Methods Three doses of SSP extraction were administered orally. Evaluations of motor and balance abilities and detection of brain edema were performed, and BBB permeability were assessed by Evans blue (EB) staining. Primary brain microvascular endothelial cells (BMECs) were subjected to oxygen-glucose deprivation, and incubated with high dose SSP drug-containing serum and VIP-antagonist respectively. Transendothelial electrical resistance (TEER) assay and Tetramethylrhodamine isothiocyanate (TRITC)-dextran (4.4 kDa) and fluorescein isothiocyanate (FITC)-dextran (70 kDa) were used to evaluate the features of paracellular junction. Western blot detected the expressions of Claudin-5, ZO-1, Occludin and VE-cadherin, matrix metalloproteinase (MMP) 2/9 and VIP receptors 1/2, and immunofluorescence staining tested VIP and Claudin-5 expressions. Results Our results show that SSP significantly reduces EB infiltration in dose-dependent manner in vivo and attenuates TRITC- dextran and FITC-dextran diffusion in vitro, and strengthens endothelial junctional complexes as represented by decreasing Claudin-5, ZO-1, Occludin and VE-cadherin degradations and MMP 2/9 expression, as well as promoting TEER in BMECs after ischemia. Moreover, it suggests that SSP notably enhances VIP and its receptors 1/2 expressions. VIP-antagonist exacerbates paracellular barrier of BMECs, while the result is reversed after incubation with high dose SSP drug-containing serum. Additionally, SSP also improve brain edema and motor and balance abilities after ischemic stroke. Conclusions we firstly demonstrate that the ameliorated efficacy of SSP on BBB permeability is related to the enhancements of VIP and its receptors, suggesting SSP might be an effective therapeutic agent on maintaining BBB integrity post-stroke.

Salvianolate lyophilized injection (SLI) strengthens blood-brain barrier function related to ERK1/2 and Akt signaling pathways.

The salvianolate lyophilized injection (SLI) has been widely used for the treatment of acute cerebral infarction; however, the molecular mechanism of how it strengthens blood brain barrier (BBB) function is not well understood. Here, we investigated the effects of SLI on BBB function in bEnd.3 cells as well as in rats. In oxygen glucose deprivation/reoxygenation (OGD/R)-damaged bEnd.3 cells, SLI increased transepithelial electrical resistance and decreased sodium fluorescein flux. SLI-treated cells showed increased expression of tight junction proteins, including Zonula occludens-1 (ZO-1), Claudin-5 and Occludin. Furthermore, SLI led to the decrease of phosphorylation of ERK1/2, p38, and Akt. Using selective inhibitors, we found that the positive effects of SLI on barrier function were abolished in cells in which ERK1/2 and Ak signaling were inhibited. Moreover, in MCAO model rats, SLI effectively alleviated brain leakage of Evans blue, increased brain tissue ZO-1 expression and inhibited phosphorylation of ERK1/2 and Akt. Overall, these data suggest that SLI strengthens BBB function was interrelated ERK1/2 and Akt signaling pathways in cerebral vascular diseases.

Puerariae Lobatae Radix with chuanxiong Rhizoma for treatment of cerebral ischemic stroke by remodeling gut microbiota to regulate the brain-gut barriers.

The combination of Puerariae Lobatae Radix (PLR) and Chuanxiong Rhizoma (CXR) is commonly used to treat cerebrovascular diseases. This work aimed to clarify the mechanisms of their action in treating cerebral ischemic stroke from the perspective of gut microecology. The PLR and CXR combination effectively improved the neurological function, reduced the cerebral infarction and relieved the complications of cerebral ischemic stroke, including dyslipidemia, increased blood viscosity and thrombotic risk. Cerebral ischemic stroke triggered gut microbial disturbances by enriching pathogens and opportunistic microorganisms, including Bacteroides, Escherichia_Shigella, Haemophilus, Eubacterium_nodatum_group, Collinsella, Enterococcus, Proteus, Alistipes, Klebsiella, Shuttleworthia and Faecalibacterium. Cerebral ischemic stroke also increased the intestinal permeability, disrupted the gut barrier and caused intestinal microbial translocation. Occludin, claudin-5 and ZO-1 levels in the brain-gut barriers showed a high positive correlation. However, the combination remodeled the gut microecology by modulating endogenous bacteria whose effects may mitigate cerebral damage, such as Alloprevotella, Ruminococcaceae, Oscillospira, Lachnospiraceae_NK4B4_group, Akkermansia and Megasphaera, protected the brain-gut barriers by increasing claudin-5 and ZO-1 levels; and weakened the gut microbiota translocation by decreasing diamine oxidase, lipopolysaccharide and d-lactate. Although nimodipine effectively reduced the cerebral infarction, it did not relieve the gut microbiota dysbiosis and instead aggravated the gut barrier disruption and microbiota translocation. In conclusion, cerebral ischemic stroke caused gut microbiota dysbiosis, increased intestinal permeability, disrupted the gut barrier and triggered gut microbiota translocation. The PLR and CXR combination was an effective treatment for cerebral ischemic stroke that relieved the gut microbiota dysbiosis and brain-gut barriers disruption.

One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood-brain barrier permeability in fed or overnight fasted rats.

Objectives: The hypothalamus lies adjacent to the third ventricle and is in close proximity with the median eminence (ME), a circumventricular organ with an incomplete blood-brain barrier (BBB) which controls direct entry of nutrients into the brain. The blood-CSF barrier of the hypothalamus shows dynamic changes upon neuroendocrine events and adjusts permeability with the tight junction (TJ) complex. It has been shown that chronic exposure to a high-fat diet (HFD) affects BBB permeability. HFD also induces leptin resistance and alters neuropeptide expression in the arcuate nucleus (Arc) of the hypothalamus starting early during overnutrition. We hypothesized altered integrity of the BBB to occur after exposing rats to a free-choice high-fat high-sugar (fcHFHS) diet for 1 week. Methods: We measured diffusion of Evans blue dye over the ME and assessed expression of the TJ proteins ZO-1, claudin-5, and occludin in the tanycytic wall of the third ventricle. Furthermore, we assessed protein expression of glucose transporter 1 (GLUT-1), which is highly expressed in the Arc-ME complex and facilitates glucose transport over the BBB. Results: fcHFHS-fed rats increased caloric intake compared to control, however, there was no effect of the fcHFHS diet on permeability of the BBB, nor changes in protein expression of tight TJ proteins or GLUT-1. Fasting acutely affects the BBB and we hypothesized that exposure to the fcHFHS diet affects the BBB differently compared to chow after fasting. We did not, however, find any differences in Evans blue diffusion nor protein expression between chow- and fcHFHS-fed rats when fasted overnight. Conclusions: We conclude that short-term consumption of a fcHFHS diet does not change permeability or diffusion in the hypothalamus barrier in ad libitum fed or fasted rats.

The sonic hedgehog pathway mediates Tongxinluo capsule-induced protection against blood-brain barrier disruption after ischaemic stroke in mice.

Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.

[Protective effect of catalpolon destruction of tight junctions of high glucose induced BMECs].

This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, α-actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury.

Angioedema and Hemorrhage After 4.5-Hour tPA (Tissue-Type Plasminogen Activator) Thrombolysis Ameliorated by T541 via Restoring Brain Microvascular Integrity.

Background and Purpose- tPA (tissue-type plasminogen activator) is the only recommended intravenous thrombolytic agent for ischemic stroke. However, its application is limited because of increased risk of hemorrhagic transformation beyond the time window. T541 is a Chinese compound medicine with potential to attenuate ischemia and reperfusion injury. This study was to explore whether T541-benefited subjects underwent tPA thrombolysis extending the time window. Methods- Male C57BL/6 N mice were subjected to carotid artery thrombosis by stimulation with 10% FeCl3 followed by 10 mg/kg tPA with/without 20 mg/kg T541 intervention at 4.5 hours. Thrombolysis and cerebral blood flow were observed dynamically until 24 hours after drug treatment. Neurological deficit scores, brain edema and hemorrhage, cerebral microvascular junctions and basement membrane proteins, and energy metabolism in cortex were assessed then. An in vitro hypoxia/reoxygenation model using human cerebral microvascular endothelial cells was used to evaluate effect of T541 on tight junctions and F-actin in the presence of tPA. Results- tPA administered at 4.5 hours after carotid thrombosis resulted in a decrease in thrombus area and survival rate, whereas no benefit on cerebral blood flow. Study at 24 hours after tPA administration revealed a significant angioedema and hemorrhage in the ischemia hemisphere, a decreased expression of junction proteins claudin-5, zonula occludens-1, occludin, junctional adhesion molecule-1 and vascular endothelial cadherin, and collagen IV and laminin. Meanwhile, ADP/ATP, AMP/ATP, and ATP5D (ATP synthase subunit) expression and activities of mitochondria complex I, II, and IV declined, whereas malondialdehyde and 8-Oxo-2'-deoxyguanosine increased and F-actin arrangement disordered. All the insults after tPA treatment were attenuated by addition of T541 dose dependently. Conclusions- The results suggest T541 as a potential remedy to attenuate delayed tPA-related angioedema and hemorrhage and extend time window for tPA treatment. The potential of T541 to upregulate energy metabolism and protect blood-brain barrier is likely attributable to its effects observed.

Jia-Jian-Di-Huang-Yin-Zi decoction exerts neuroprotective effects on dopaminergic neurons and their microenvironment.

As a classical prescription of Traditional Chinese medicine, the Jia-Jian-Di-Huang-Yin-Zi (JJDHYZ) decoction has long been used to treat movement disorders. The present study evaluated the effects of JJDHYZ on dopaminergic (DA) neurons and their survival-enhancing microenvironment as well as the possible mechanisms involved using a mouse model of Parkinson's disease. In MPTP-lesioned mice, a high dosage of JJDHYZ (34 g/kg/day) attenuated the loss of DA neurons, reversed the dopamine depletion, and improved the expression of glial-derived neurotrophic factor (GDNF) compared to the untreated model group. JJDHYZ also protected the ultrastructure of the blood-brain barrier (BBB) and tight junction proteins by inhibiting the activation of microglia and astrocytes besides the increase in three types of matrix metalloproteinases in the substantia nigra. In conclusion, the JJDHYZ-high dosage (JJDHYZ-H) group exhibited the neuroprotection of DA neurons, and the underlying mechanism may be related to the survival-enhancing microenvironment of the DA neurons.

Contribution of Borneolum syntheticum to the Intervention Effect of Liuwei Dihuang Pill () on Experimental Retinal Degeneration.

OBJECTIVE: To observe the contribution of Borneolum syntheticum to the intervention effect of Liuwei Dihuang Pill (, LDP) on experimental retinal degeneration, and initially investigate the mechanism of Borneolum syntheticum as meridian-lead-in drug. METHODS: A total of 180 sodium iodateinduced retinital degeneration rats were randomly divided into three groups, including distilled water group, LDP group, and LDP+Borneolum syntheticum (LDP+BS) group. Twenty normal rats were fed regularly without any treatment as normal control. On day 7 and 14 after treatment, histopathological study and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) test were performed to evaluate the retinopathy. Claudin-5 expression at blood-retina barrier (BRB) was detected by Western blot at different time points from 0.5 to 8 h after gavage. RESULTS: On day 7 and 14 after treatment, the retinal lesion grades were significantly different among the three groups (P<0.05). The grade in the LDP+BS group was significantly less than the LDP and distilled water groups (both P<0.05), no significant difference was observed between the LDP and distilled water groups (P>0.05). The apoptosis rates in the LDP+BS group was significantly less than the distilled water and LDP groups (both P<0.05), while there was no significant difference between LDP and distilled water groups (P>0.05). Expression of claudin-5 in LDP+BS group was significantly less than the other two groups at 0.5, 1 and 2 h after gavage (P<0.05). There was no apparent difference among the three groups at 4 and 8 h after gavage (P>0.05). CONCLUSION: Borneolum syntheticum could strengthen the effect of LDP on experimental retinal degeneration, indicated that Borneolum syntheticum might play the role of meridian-lead-in drug in the formula. The mechanism may be due to Borneolum syntheticum could promote the physiologically openness of bloodretina barrier through transiently affecting the expression of claudin-5.