Arsenic and Chromium Induced Toxicity on Zebrafish Kidney: Mixture Effects on Oxidative Stress and Involvement of Nrf2-Keap1-ARE, DNA Repair, and Intrinsic Apoptotic Pathways.

In polluted water, cooccurrences of two carcinogens, arsenic (As) and chromium (Cr), are extensively reported. Individual effects of these heavy metals have been reported in kidney of fishes, but underlying molecular mechanisms are not well established. There is no report on combined exposure of As and Cr in kidney. Thus, the present study investigated and compared individual and combined effects of As and Cr on zebrafish (Danio rerio) kidney treating at their environmentally relevant concentrations for 15, 30, and 60 days. Increased ROS levels, lipid peroxidation, GSH level, and decreased catalase activity implied oxidative stress in treated zebrafish kidney. Damage in histoarchitecture in treated groups was also noticed. The current study involved gene expression study of Nrf2, an important transcription factor of cellular stress responses along with its negative regulator Keap1 and downstream antioxidant genes nqo1 and ho1. Results indicated activation of Nrf2-Keap1 pathway after combined exposure. Expression pattern of ogg1, apex1, polb, and creb1 revealed the inhibition of base excision repair pathway in treatments. mRNA expression of tumor suppressor genes p53 and brca2 was also altered. Expressional alteration in bax, bcl2, caspase9, and caspase 3 indicated apoptosis (intrinsic pathway) induction, which was maximum in combined group. Inhibition of DNA repair and induction of apoptosis indicated that the activated antioxidant system was not enough to overcome the damage caused by As and Cr. Overall, this study revealed additive effects of As and Cr in zebrafish kidney after chronic exposure focusing cellular antioxidant and DNA damage responses.

Development of a Nanomarker for In Vivo Monitoring of Dopamine in Plants.

Dopamine, alongside norepinephrine and epinephrine, belongs to the catecholamine group, widely distributed across both plant and animal kingdoms. In mammals, these compounds serve as neurotransmitters with roles in glycogen mobilization. In plants, their synthesis is modulated in response to stress conditions aiding plant survival by emitting these chemicals, especially dopamine that relieves their resilience against stress caused by both abiotic and biotic factors. In present studies, there is a lack of robust methods to monitor the operations of dopamine under stress conditions or any adverse situations across the plant's developmental stages from cell to cell. In our study, we have introduced a groundbreaking approach to track dopamine generation and activity in various metabolic pathways by using the simple nitrogen and sulfur co-doped carbon quantum dots (N, S-CQDs). These CQDs exhibit dominant biocompatibility, negligible toxicity, and environmentally friendly characteristics using a quenching process for fluorometric dopamine detection. This innovative nanomarker can detect even small amounts of dopamine within plant cells, providing insights into plant responses to strain and anxiety. Confocal microscopy has been used to corroborate this occurrence and to provide visual proof of the process of binding dopamine with these N, S-CQDs inside the cells.

RNA-Seq time-course analysis of neural precursor cell transcriptome in response to herpes simplex Virus-1 infection.

The neurogenic niches within the central nervous system serve as essential reservoirs for neural precursor cells (NPCs), playing a crucial role in neurogenesis. However, these NPCs are particularly vulnerable to infection by the herpes simplex virus 1 (HSV-1). In the present study, we investigated the changes in the transcriptome of NPCs in response to HSV-1 infection using bulk RNA-Seq, compared to those of uninfected samples, at different time points post infection and in the presence or absence of antivirals. The results showed that NPCs upon HSV-1 infection undergo a significant dysregulation of genes playing a crucial role in aspects of neurogenesis, including genes affecting NPC proliferation, migration, and differentiation. Our analysis revealed that the CREB signaling, which plays a crucial role in the regulation of neurogenesis and memory consolidation, was the most consistantly downregulated pathway, even in the presence of antivirals. Additionally, cholesterol biosynthesis was significantly downregulated in HSV-1-infected NPCs. The findings from this study, for the first time, offer insights into the intricate molecular mechanisms that underlie the neurogenesis impairment associated with HSV-1 infection.

Chronic low-dose chromium VI exposure induces oxidative stress and apoptosis with altered expressions of DNA repair genes and promoter hypermethylation in the liver of Swiss albino mice.

The present investigation dealt with harmful effects of hexavalent chromium (Cr [VI]) on liver of Swiss albino mice. This variant exhibited cytotoxicity, mutagenicity, and carcinogenicity. Our study focused on elucidating the hepatotoxic effects of chronic low-dose exposure to Cr (VI) (2, 5, and 10 ppm) administered via drinking water for 4 and 8 months. The observed elevation in SGPT, ALP, and SGOT and increased oxidative stress markers unequivocally confirmed the severe disruption of liver homeostasis at these low treatment doses. Noteworthy alterations in histoarchitecture, body weight, and water intake provided further evidences of the harmful effects of Cr (VI). Production of reactive oxygen species (ROS) during metabolism led to DNA damages. Immunohistochemistry and qRT-PCR analyses revealed that chronic low-dose exposure of Cr (VI) induced apoptosis in liver tissue. Our study exhibited alterations in the expression pattern of DNA repair genes (Rad51, Mutyh, Mlh1, and Ogg1), coupled with promoter hypermethylation of Mutyh and Rad51, leading to transcriptional inhibition. Our findings underscored the potential of low-dose Cr (VI) exposure on hepatotoxicity by the intricate interplay between apoptosis induction and epigenetic alterations of DNA repair genes.

Arsenic and chromium induced hepatotoxicity in zebrafish (Danio rerio) at environmentally relevant concentrations: Mixture effects and involvement of Nrf2-Keap1-ARE pathway.

Arsenic (As) and chromium (Cr), two well-known cytotoxic and carcinogenic metals are reported to coexist in industrial effluents and groundwater. Their individual toxicities have been thoroughly studied but the combined effects, especially the mechanism of toxicity and cellular stress response remain unclear. Considering co-exposure as a more realistic scenario, current study compared the individual and mixture effects of As and Cr in the liver of zebrafish (Danio rerio). Fish were exposed to environmentally relevant concentrations of As and Cr for 15, 30 and 60 days. ROS generation, biochemical stress parameters like lipid peroxidation, reduced glutathione content, catalase activity and histological alterations were studied. Results showed increase in ROS production, MDA content and GSH level; and vicissitude in catalase activity as well as altered histoarchitecture, indicating oxidative stress conditions after individual and combined exposure of As and Cr which were additive in nature. This study also included the expression of Nrf2, the key regulator of antioxidant stress responses and its nuclear translocation. Related antioxidant and xenobiotic metabolizing enzyme genes like keap1, nqo1, ho1, mnsod and cyp1a were also studied. Overall results indicated increased nrf2, nqo1, ho1, mnsod expression at all time points and increased cyp1a expression after 60 days exposure. Emphasizing on the Nrf2-Keap1 pathway, this study exhibited additive or sometimes synergistic effects of As and Cr in zebrafish liver.

Lead induced genotoxicity and hepatotoxicity in zebrafish (Danio rerio) at environmentally relevant concentration: Nrf2-Keap1 regulated stress response and expression of biomarker genes.

Genotoxic and hepatotoxic potentials of Pb at an environmentally relevant concentration (5 ppm) in zebrafish were investigated in the present study. Erythrocytic nuclear abnormality tests revealed the increased frequencies of abnormal erythrocytes after Pb exposure, indicating a strong genotoxic potential of Pb. Multiple stress-related parameters were further evaluated in liver, the major detoxifying organ. Pb caused increased production of ROS, which in turn caused severe oxidative stress. As a result, lipid peroxidation was increased, whereas reduced glutathione level and catalase activity was decreased. Alterations in liver histoarchitecture also served as evidence of Pb-induced hepatotoxicity. Pb-induced ROS stress triggered upregulation of Nrf2, Nqo1, Ho1; downregulation of Keap1, and altered mRNA expressions of Mn-sod, Cu/Zn-sod, gpx1, cyp1a, ucp2 suggesting involvement of Nrf2-Keap1-ARE signaling in cellular defence. Nrf2-keap1 is a sensitive biomarker of Pb-induced ROS stress. Overexpression of Hsp70 and other genes in hepatocytes might help cell survival under oxidative stress generation.

Environmentally relevant lead alters nuclear integrity in erythrocytes and generates oxidative stress in liver of Anabas testudineus: Involvement of Nrf2-Keap1 regulation and expression of biomarker genes.

Genotoxic and hepatotoxic effects of lead (Pb) on a freshwater fish, climbing perch (Anabas testudineus) were studied at an environmentally relevant concentration (43.3 ppm). The genotoxic potential of Pb was confirmed by micronucleus study, with increased frequencies of erythrocytic nuclear alterations like lobed, blebbed, notched, fragmented, and micronuclei were observed in erythrocytes in treated groups as compared to control. Inorganic Pb induces oxidative stress which is a consequence of elevated level of Reactive Oxygen Species. Hepatotoxicity was assessed both by the oxidative stress and cellular responses that emerged due to the toxic assault of Pb in the liver, the most important detoxifying organ. Upregulation of xenobiotic metabolizing enzyme like catalase was evident after 15, 30, and 90 days of exposure, and a profound effect was observed on 30th days. The level of lipid peroxidation and reduced glutathione was increased after Pb exposure. Histoarchitectural damages of liver were distinctly evident in treated fish. Western blot analysis confirmed the expressional alterations of stress-responsive marker proteins like Nrf2, Keap1, Hsp70, and Nqo1. Pb exposure resulted in increased expression of Hsp70, Nrf2, and Nqo1, whereas Keap1 was downregulated, suggesting the involvement of Nrf2-Keap1 regulation as a cytoprotective mechanism against Pb toxicity.

Efficient delivery of methotrexate to MDA-MB-231 breast cancer cells by a pH-responsive ZnO nanocarrier.

Methotrexate (MTX), an efficient chemotherapy medication is used in treating various malignancies. However, the breast cancer cell line MDA-MB-231 has developed resistance to it due to low levels of the MTX transport protein, and reduced folate carrier (RFC), making it less effective against these cancer cells. Here we designed a very simple, biocompatible, and non-toxic amine-capped ZnO quantum dots to overcome the MTX resistance on the MDA-MB-231 breast cancer cell line. The QD was characterized by HRTEM, DLS EDX, FT-IR, UV-Vis, and Fluorescence spectroscopy. MTX loading onto the QD was confirmed through fluorescence and UV-Vis spectroscopy. Additionally, extensive confocal microscopic investigations were carried out to determine whether the MTX was successfully released on the MDA-MB-231 cell line. It was discovered that QD is a better pH-responsive delivery system than the previous ones because it successfully delivers MTX to the MDA-MB-231 at a higher rate on an acidic pH than it does at a physiological pH. QD also has anticancer activity and can eradicate cancer cells on its own. These factors make the QD to be an effective pH-responsive delivery system that can improve the efficacy of the medication in therapeutic diagnosis.

Direct fluorescence labelling of NO inside plant cells.

Nitric oxide (NO) plays a key role in regulating plant growth, enhances nutrient uptake, and activates disease and stress tolerance mechanisms in most plants. NO is marked as a potential tool for improving the yield and quality of horticultural crop species. Research on NO in plant species can provide an abundance of valuable information regarding this. Hence, we have prepared a simple chemosensor (NPO) for the detection of endogenous NO in chickpea saplings. NPO selectively interacts with NO as determined through a chemodosimetric method to clearly show both the colorimetric and fluorometric changes. After the interaction with NO, the colorless NPO turns yellow as observed by the naked eye and shows bright cyan-blue fluorescence under a UV lamp. The 1 : 1 stoichiometric ratio between NPO and NO is determined from Job's plot resulting in a stable diazeniumdiolate product. The interaction mechanism is well established by absorption, fluorescence titration, NMR titration, HRMS, and DFT calculations. This method has successfully been employed in the plant's root and stem systems to label NO. Confocal microscopy images might help us to understand the endogenous NO generation and the mechanism that happens inside plant tissues.

Differential detection of aspartic acid in MCF-7 breast cancer cells.

Aspartic acid is a non-essential amino acid obtained in the neuroendocrine tissues of vertebrates and invertebrates. Aspartic acid, a major excitatory neurotransmitter in the mammalian central nervous system, plays a key role in memory and acts in many other normal and abnormal physiological processes. In this work, we have developed an efficient chemosensor (PCF) based on the pyridine-carbazole moiety for the differential detection of aspartic acid in biological systems. PCF has a strong binding affinity towards aspartic acid, with a detection limit in the nanomolar range. The binding stoichiometry of aspartic aid and PCF was obtained as 1 : 1 from a Jobs plot analysis. Furthermore, the efficacy of PCF has been successfully demonstrated in in vitro experiments in MCF-7 breast cancer cells.

Chronic exposure to environmentally relevant concentration of fluoride impairs osteoblast's collagen synthesis and matrix mineralization: Involvement of epigenetic regulation in skeletal fluorosis.

Globally, 200 million people are suffering from toxic manifestations of Fluoride(F), dental and skeletal fluorosis; unfortunately, there is no treatment. To unravel the pathogenesis of skeletal fluorosis, we established fluorosis mice by treating environmentally relevant concentration of F (15 ppm NaF) through drinking water for 4 months. As in skeletal fluorosis, locomotor disability, crippling deformities occur and thus, our hypothesis was F might adversely affects collagen which gives the bone tensile strength. This work inevitably had to be carried out on osteoblast cells, responsible for synthesis, deposition, and mineralization of bone matrix. Isolated osteoblast cells were confirmed by ALP activity and mineralized nodules formation. Expression of collagen Col1a1, Col1a2, COL1A1 was significantly reduced in treated mice. Further, a study revealed the involvement of epigenetic regulation by promoter hypermethylation of Col1a1; expressional alterations of transcription factors, calcium channels and other genes e.g., Cbfa-1, Tgf-ß1, Bmp1, Sp1, Sp7, Nf-Kb p65, Bmp-2, Bglap, Gprc6a and Cav1.2 are associated with impairment of collagen synthesis, deposition and decreased mineralization thus, enfeebling bone health. This study indicates the possible association of epigenetic regulation in skeletal fluorosis. However, no association was found between polymorphisms in the Col1a1 (RsaI, HindIII) and Col1a2 (RsaI, HindIII) genes with fluorosis in mice.

Ecotoxicology of hexavalent chromium in fish: An updated review.

Chromium (Cr) is prevalently found in trivalent and hexavalent forms. Though the former is toxicologically benign due to its poor cellular permeability, hexavalent chromium i.e. Cr [VI] crosses the biological membrane and induces toxic effects in organisms. While Cr [VI] toxicity in humans is a subject of occupational exposure at industries involved in ferrochrome production, leather tanning, textile dyeing etc., aquatic abundance of Cr [VI] due to discharge of Cr-laden effluents by these industries lead to extensive toxicity in piscine species. The present review aims to discuss the mode of Cr [VI] entry in fish and the several inimical effects that it imparts on fish health. Such effects have been reported in various studies through behavioral, hormonal and hematological alterations. Bio-accumulation of Cr [VI] in vital organs and subsequent perturbation of the oxidative homeostasis leads to organotoxic effects like changes in organo-somatic indices and histo-architecture. At cellular level, Cr [VI] induced genotoxicity is often found to trigger cellular demise including apoptosis. This review also highlights the stress response in fish against Cr [VI] induced toxicity that is mediated through the expressional alteration of a myriad of anti-oxidant and xenobiotic-metabolizing proteins which is, in turn, a function of activated transcription programs including the Nrf2-ARE pathway.

NAT8L mRNA oxidation is linked to neurodegeneration in multiple sclerosis.

RNA oxidation has been implicated in neurodegeneration, but the underlying mechanism for such effects is unclear. Extensive RNA oxidation occurs within the neurons in multiple sclerosis (MS) brains. Here, we identified selectively oxidized mRNAs in neuronal cells that pertained to neuropathological pathways. N-acetyl aspartate transferase 8 like (NAT8L) is one such transcript, whose translation product enzymatically synthesizes N-acetyl aspartic acid (NAA), a neuronal metabolite important for myelin synthesis. We reasoned that impediment of translation of an oxidized NAT8L mRNA will result in a reduction in its cognate protein, thus lowering the NAA level. This hypothesis is supported by our studies on cells, an animal model, and postmortem human MS brain. Reduced brain NAA level hampers myelin integrity making neuronal axons more susceptible to damage, which contributes to MS neurodegeneration. Overall, this work provides a framework for a mechanistic understanding of the link between RNA oxidation and neurodegeneration.

Brain-derived neurotrophic factor (BDNF) epigenomic modifications and brain-related phenotypes in humans: A systematic review.

Epigenomic modifications of the brain-derived neurotrophic factor (BDNF) gene have been postulated to underlie the pathogenesis of neurodevelopmental, psychiatric, and neurological conditions. This systematic review summarizes current evidence investigating the association of BDNF epigenomic modifications (DNA methylation, non-coding RNA, histone modifications) with brain-related phenotypes in humans. A novel contribution is our creation of an open access web-based application, the BDNF DNA Methylation Map, to interactively visualize specific positions of CpG sites investigated across all studies for which relevant data were available. Our literature search of four databases through September 27, 2021 returned 1701 articles, of which 153 met inclusion criteria. Our review revealed exceptional heterogeneity in methodological approaches, hindering the identification of clear patterns of robust and/or replicated results. We summarize key findings and provide recommendations for future epigenomic research. The existing literature appears to remain in its infancy and requires additional rigorous research to fulfill its potential to explain BDNF-linked risk for brain-related conditions and improve our understanding of the molecular mechanisms underlying their pathogenesis.

Reliable fluorescence technique to detect the antibiotic colistin, a possible environmental threat due to its overuse.

Colistin, considered a drug of last resort as it is effective towards multidrug-resistant Gram-negative bacterial infections. Oral administration of colistin in the poultry industry is a common practice, not only to prevent and reduce bacterial infections, but also as a rapid-growth promoter. Long-term exposure to any antibiotic will eventually lead to the development of bacterial resistance towards all antibiotics through various mechanisms in the physiological system and environment. Chicken is the most consumed source of animal protein for humans throughout the world. In addition, the manure of poultry, containing traces of the used antibiotics, is being used in farming. Exposure to excess amounts of colistin causes a great concern not only to the humans but to the environment as a whole. In the present contribution, colistin has been detected in chicken hepatocyte cells through in vivo confocal microscopy. In addition, the amount of colistin in the chicken excrements has been estimated. A simple chemosensor NAF, a dye-based on napthaldehyde furfural, was developed for the detection of colistin, supplemented with experimental evidence and theoretical calculations.

Attenuated cell-cycle division protein 2 and elevated mitotic roles of polo-like kinase 1 characterize deficient myoblast fusion in peripheral arterial disease.

INTRODUCTION: We propose that MuSC-derived myoblasts in PAD have transcriptomic differences that can highlight underlying causes of ischemia-induced myopathy. METHODS: Differentiation capacity among perfused and ischemic human myoblasts was compared. Following next generation sequencing of mRNA, Ingenuity Pathway Analysis (IPA) was performed for canonical pathway enrichment. Live cell imaging and immunofluorescence were performed to determine myocyte fusion index and protein expression based on insights from IPA, specifically concerning cell cycle regulators including cell-division cycle protein 2 (CDC2) and polo-like kinase 1 (PLK1). RESULTS: Ischemic myoblasts formed attenuated myotubes indicative of reduced fusion. Additionally, myoblasts from ischemic segments showed significant differences in canonical pathways associated with PLK1 (upregulated) and G2/M DNA damage checkpoint regulation (downregulated). PLK1 inhibition with BI2536 did not affect cell viability in any group over 24 h but deterred fusion more significantly in PAD myoblasts. Furthermore, PLK1 inhibition reduced the expression of checkpoint protein CDC2 in perfused but not ischemic cells. CONCLUSION: Differentiating myoblasts derived from ischemic muscle have significant differences in gene expression including those essential to DNA-damage checkpoint regulation and cell cycle progress. DNA-damage checkpoint dysregulation may contribute to myopathy in PAD.

NF-κB Signaling-Mediated Activation of WNK-SPAK-NKCC1 Cascade in Worsened Stroke Outcomes of Ang II-Hypertensive Mice.

BACKGROUND: Worsened stroke outcomes with hypertension comorbidity are insensitive to blood pressure-lowering therapies. In an experimental stroke model with comorbid hypertension, we investigated causal roles of ang II (angiotensin II)-mediated stimulation of the brain WNK (with no lysine [K] kinases)-SPAK (STE20/SPS1-related proline/alanine-rich kinase)-NKCC1 (Na-K-Cl cotransporter) complex in worsened outcomes. METHODS: Saline- or ang II-infused C57BL/6J male mice underwent stroke induced by permanent occlusion of the distal branches of the middle cerebral artery. Mice were randomly assigned to receive either vehicle dimethyl sulfoxide/PBS (2 mL/kg body weight/day, IP), a novel SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (ZT-1a' 5 mg/kg per day, IP) or a NF-κB (nuclear factor-κB) inhibitor TAT-NBD (transactivator of transcription-NEMO-binding domain' 20 mg/kg per day, IP). Activation of brain NF-κB and WNK-SPAK-NKCC1 cascade as well as ischemic stroke outcomes were examined. RESULTS: Stroke triggered a 2- to 5-fold increase of WNK (isoforms 1, 2, 4), SPAK/OSR1 (oxidative stress-responsive kinase 1), and NKCC1 protein in the ang II-infused hypertensive mouse brains at 24 hours after stroke, which was associated with increased nuclear translocation of phospho-NF-κB protein in the cortical neurons (a Pearson correlation r of 0.77, P<0.005). The upregulation of WNK-SPAK-NKCC1 cascade proteins resulted from increased NF-κB recruitment on Wnk1, Wnk2, Wnk4, Spak, and Nkcc1 gene promoters and was attenuated by NF-κB inhibitor TAT-NBD. Poststroke administration of SPAK inhibitor ZT-1a significantly reduced WNK-SPAK-NKCC1 complex activation, brain lesion size, and neurological function deficits in the ang II-hypertensive mice without affecting blood pressure and cerebral blood flow. CONCLUSIONS: The ang II-induced stimulation of NF-κB transcriptional activity upregulates brain WNK-SPAK-NKCC1 cascade and contributes to worsened ischemic stroke outcomes, illustrating the brain WNK-SPAK-NKCC1 complex as a therapeutic target for stroke with comorbid hypertension.

Environmentally Relevant Hexavalent Chromium Disrupts Elemental Homeostasis and Induces Apoptosis in Zebrafish Liver.

Although hexavalent chromium Cr [VI] is known as a toxicant in the aquatic environment, its effect in low, environmentally relevant concentration (ERC; 2 mg L-1) is less characterized. Against this backdrop, the effects of Cr [VI] in ERC on zebrafish liver has been investigated in this study. Fluorescence microscopy and gel electrophoresis detected excess DNA damage and cell death via apoptosis in 2 mg L-1 Cr [VI]-treated fish when compared with that of control. Besides, there were transcriptional activations of p53, Bax, Caspase 9 and Caspase 3 genes but downregulation of Bcl2 gene in the treated group, confirming the apoptotic pathway. Energy dispersive X-ray fluorescence (EDXRF) data showed significant (p < 0.05) increase in hepatic content of Cr, selenium, iron, manganese, calcium, sulfur and magnesium but depletion of zinc, copper and cobalt in the treated group. Collectively, the study shows that even a low, ERC of Cr [VI] is toxic to the zebrafish as it elicited marked apoptosis in the hepatocytes and altered the liver elemental profile.

Toxic and carcinogenic effects of hexavalent chromium in mammalian cells in vivo and in vitro: a recent update.

Chromium VI (Cr (VI)) can cross cell membranes readily and causes the formation of Cr-DNA adducts, genomic damages, elevation of reactive oxygen species (ROS) and alteration of survival signaling pathways, as evidenced by the modulation in p53 signaling pathway. Mammals, including humans are exposed to Cr, including Cr (VI), frequently through inhalation, drinking water, and food. Several studies demonstrated that Cr (VI) induces cellular death through apoptosis and autophagy, genotoxicity, functional alteration of mitochondria, endocrine and reproductive impairments. In the present review, studies on deleterious effects of Cr (VI) exposure to mammalian cells (in vivo and in vitro) have been documented. Special attention is paid to the underlying molecular mechanism of Cr (VI) toxicity.

Attenuating vascular stenosis-induced astrogliosis preserves white matter integrity and cognitive function.

BACKGROUND: Chronic cerebral hypoperfusion (CCH) causes white matter damage and cognitive impairment, in which astrogliosis is the major pathology. However, underlying cellular mechanisms are not well defined. Activation of Na+/H+ exchanger-1 (NHE1) in reactive astrocytes causes astrocytic hypertrophy and swelling. In this study, we examined the role of NHE1 protein in astrogliosis, white matter demyelination, and cognitive function in a murine CCH model with bilateral carotid artery stenosis (BCAS). METHODS: Sham, BCAS, or BCAS mice receiving vehicle or a selective NHE1 inhibitor HOE642 were monitored for changes of the regional cerebral blood flow and behavioral performance for 28 days. Ex vivo MRI-DTI was subsequently conducted to detect brain injury and demyelination. Astrogliosis and demyelination were further examined by immunofluorescence staining. Astrocytic transcriptional profiles were analyzed with bulk RNA-sequencing and RT-qPCR. RESULTS: Chronic cerebral blood flow reduction and spatial working memory deficits were detected in the BCAS mice, along with significantly reduced mean fractional anisotropy (FA) values in the corpus callosum, external capsule, and hippocampus in MRI DTI analysis. Compared with the sham control mice, the BCAS mice displayed demyelination and axonal damage and increased GFAP+ astrocytes and Iba1+ microglia. Pharmacological inhibition of NHE1 protein with its inhibitor HOE642 prevented the BCAS-induced gliosis, damage of white matter tracts and hippocampus, and significantly improved cognitive performance. Transcriptome and immunostaining analysis further revealed that NHE1 inhibition specifically attenuated pro-inflammatory pathways and NADPH oxidase activation. CONCLUSION: Our study demonstrates that NHE1 protein is involved in astrogliosis with pro-inflammatory transformation induced by CCH, and its blockade has potentials for reducing astrogliosis, demyelination, and cognitive impairment.