Compartment-specific activation of kinin B1 and B2 receptors drives the production of vasoactive and inflammatory mediators during SARS-CoV-2 infection.

Pregabalin, a first-line treatment for neuropathic pain, binds to the voltage-gated calcium channel auxiliary subunit alpha2delta-1, reducing neurotransmitter release. Transdermal pregabalin has also shown analgesic effects in neuropathic mouse models. However, its effects on epidermal cells remain unclear. We aimed to investigate the action of pregabalin on the human Merkel cell line (MC). We used the cultured human Merkel cell line MCC 14/2 to measure intracellular free calcium concentration ([Ca2+]i) levels using fura-2 and to perform immunofluorescence analysis. To examine effect of pregabalin on a chronic pain condition, we applied Lys-(Des-Arg9) bradykinin (BK) for 48 hours (BK group), BK for 48 hours with pregabalin for 24 hours (BK + pregabalin group), or none (as control group) before measuring the direct mechanical stimulation-induced [Ca2+]i response in the MCs. The area under the curve (AUC) of the transient increase in [Ca2+]i was significantly increased in the BK group than that in the control group. Area under the curve did not show any significant differences between control group and BK + pregabalin group. When the Na+-Ca2+ exchanger (NCX) inhibitor KB-R7943 and SEA0400 were applied just before mechanical stimulation in the BK + pregabalin group, AUC was significantly increased compared to that in the absence of KB-R7943 and SEA0400. We could not observe any significant differences in the peak values of mechanical stimulation-induced [Ca2+]i increases among the groups. Merkel cells predominantly expressed the NCX1 isoform. These results suggested that the site of action of pregabalin to MCs is Ca2+ extrusion mechanism via NCX1.

Kinins, bioactive peptides produced through the proteolytic activity of kallikrein1, are members of the kallikrein-kinin system (KKS) and play crucial roles in regulating physiological processes such as inflammation, blood pressure, vascular permeability, and cell function and growth. In adipose tissue, bradykinin (BK) and des-Arg9-BK (DBK), produced by plasma kallikrein (KLKB1), act via their receptors B2 (B2R) and B1 (B1R), respectively. B1R predominates in preadipocytes, while B2R is expressed during adipogenesis, likely driving adipose tissue expansion and sustaining chronic low-grade inflammation, both hallmarks of obesity and its associated metabolic disorders. Obesity, a multifactorial metabolic disease, is closely linked to adipose tissue dysfunction. This dysfunction is driven by inflammation and oxidative stress, which in turn alter adipogenesis, lipolysis, and insulin and leptin signaling, contributing to obesity and its comorbidities. This review focuses on the role of the KKS in adipose tissue homeostasis and function. Evidence from animal models suggests that B1R ablation or antagonism results in a healthier phenotype, characterized by improved leptin and insulin sensitivity, increased lipid oxidation, reduced adipose hypertrophy, and diminished production of proinflammatory mediators and reactive oxygen species. Conversely, B2R activation may exert protective effects by enhancing insulin signaling and promoting glucose uptake, although its role remains incompletely understood and appears context-dependent. The KKS proposes it as a promising therapeutic target, biomarker, and prognostic indicator in anti-obesity pharmacological strategies.

C1 inhibitor: from complement system to bradykininangioedema.

Deep learning-optimized multi-enzyme hydrolysis for walnut antihypertensive peptides.

ABSTRACTObjectiveTo investigate the underlying mechanisms through which electroacupuncture (EA) at the Guanyuan (CV4) acupoint inhibits sympathetic activity and neurogenic inflammatory responses to relieve pain in rats with pelvic inflammatory disease (PID).MethodsEscherichia coli and Staphylococcus aureus were used to establish a PID rat model. EA was evaluated at frequencies of 2, 100, and 2/100 Hz, and 2/100 Hz was selected for subsequent investigation. The rats were randomly divided into the control, model, EA‐guanyuan (2/100 Hz), and EA‐nonsensitized groups (n = 6). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were assessed using von Frey filaments. Hematoxylin and eosin staining was performed to evaluate the histopathology. The tyrosine hydroxylase (TH) expression was analyzed using immunofluorescence (IF) staining. The levels of tumor necrosis factor‐α (TNF‐α), interleukin‐2 (IL‐2), transforming growth factor‐β1 (TGF‐β1), intercellular cell adhesion molecule‐1 (ICAM‐1), 5‐hydroxytryptamine receptor 3 (5‐HT3R), substance P (SP), hyaluronic acid (HA), and bradykinin (BK) were measured using an enzyme‐linked immunosorbent assay (ELISA). Western blot analysis was performed to measure the expression of 5‐HT3R, calcitonin gene‐related peptide (CGRP), HA, Kininogen 1 (KNG1), prostaglandin I2 (PGI2), and trefoil factor 2 (TFF2). Transmission electron microscopy (TEM) was used to observe synaptic connections.ResultsEA at CV4 reduced the behavioral pain score (p < 0.05), increased MWT and TWL, and alleviated uterine tissue pathological damage in rats. EA at CV4 reduced the levels of 5‐HT3R, CGRP, BK, HA, KNG1, PGI2SP, TGF‐β1, ICAM‐1, and TNF‐α, and increased IL‐2 levels (p < 0.05). Furthermore, EA at CV4 inhibited sympathetic activity by decreasing TH expression (p < 0.05). Additionally, EA at CV4 restored the synaptic connections between the pelvic nerves of the dorsal commissural neuron (DCN).ConclusionEA at CV4 alleviated the pathological damage and pain sensitization of uterine tissue in rats with PID by inhibiting sympathetic activity and neurogenic inflammatory response.

Low retinal blood flow and/or vasospasm represent major risk factors for the development of glaucomatous optic neuropathy (GON), a potentially blinding eye disease. Bradykinin (BK), a nonapeptide, is endogenously produced and released, which can cause smooth muscles to contract and relax in different tissues depending on the physiological/pathological situation and the presence or absence of vascular tone. Several reports have shown the presence of BK receptor mRNAs, and in some cases, B1- and B2-receptor proteins, in ocular tissues, including the retina. However, the function of these receptors remains to be determined, especially in retinal blood vessels. We pharmacologically characterized the ability of BK and any related peptide agonists to promote the contraction of isolated bovine posterior ciliary arteries (PCAs) in an organ bath setup using a cumulative compound addition and tension development recording process. Receptor-selective kinin agonists and subtype-selective BK receptor antagonists were utilized to define the possible heterogeneity in the functional BK receptors for PCAs. All agonist kinin peptides concentration-dependently contracted the PCA rings bi-phasically over a 5-log unit range (0.1 nM-10 μM). The relative potencies (EC50 values; n = 4-5) regarding the high-affinity receptor site were: Lys-BK = 0.9 ± 0.4 nM; Des-Arg9-BK = 0.9 ± 0.4 nM; RMP-7 = 1.1 ± 0.6 nM; Met-Lys-BK = 1.3 ± 0.5 nM; Hyp3-BK = 2.7 ± 0.5 nM; BK = 3.0 ± 0.7 nM. The low-affinity receptor site activated by these peptides mostly exhibited EC50 values ranging from 0.3 μM to 3 μM. The concentration-response curves to Des-Arg9-BK (B1-selective agonist) were shifted to the left in the presence of increasing concentrations of a B1-receptor antagonist (R715: 1-10 μM; n = 3). Similarly, WIN-64338 (a B2-receptor antagonist: 1-10 μM; n = 3) moved the BK concentration-response curves to the left. The pharmacological characteristics of BK and analog-induced contractions, and their inhibition by receptor-selective antagonists, indicated the presence of both B1- and B2-receptors, and perhaps another subtype, which mediate the PCA contractions. These results have potential implications for the involvement of heterogeneous kinin receptors, narrowing PCA diameters in vivo, restricting blood flow to the retina, causing GON, and subsequent visual impairment that can eventually cause blindness.

The kallikrein kinin system alters neuroretinal and visual responses in mice.

Background: Bradykinin (BK) is an inflammatory mediator. The degradation of labeled synthetic BK in biofluids can be used to report on the activity of angiotensin-converting enzyme (ACE) and basic carboxypeptidases N and CBP2, for which the neuropeptide is a substrate. Clinical studies have shown significant changes in the serum activity of these enzymes in patients with inflammatory diseases. Methods: Here, we investigated variation in the cleavage of dabsylated synthetic BK (DBK) in serum and the formation of the major enzymatic fragments using a thin-layer chromatography-based neuropeptide reporter assay (NRA) in a large cohort of healthy volunteers from the international human Personal Omics Profiling consortium based at Stanford University. Results: Four major outcomes were reported. First, a set of NRA reference data for the healthy population was delivered, which is important for future investigations of patient sera. Second, it was shown that the measured serum degradation capacity for DBK was significantly higher in males than in females. There was no significant correlation of the NRA results with ethnicity, body mass index or overnight fasting. Third, a batch effect was noted among sampling sites (HUPO conferences). Thus, we used subcohorts rather than the entire collection for data mining. Fourth, as the low-cost and robust NRA is sensitive to enzyme activity, it provides such a necessary quick test to eliminate degraded and/or otherwise questionable samples. Conclusions: The results reiterate the critical importance of a high level of standardization in pre-analytical sample collection and processing—most notably, sample quality should be evaluated before conducting any large and expensive omics analyses.

One of the major challenges in diagnosing various diseases, including neurological and neurodegenerative disorders, as well as carcinogenesis, is detecting unlabeled neurotransmitters. Surface-enhanced Raman spectroscopy (SERS) and surface-enhanced infrared spectroscopy (SEIRA) are promising methods for neurotransmitter biosensing and bioimaging. These methods are unique in that they are non-destructive and can identify molecular fingerprints. In this study, these methods were used to detect the following potent bradykinin (BK) antagonists: [D-Arg0,Hyp3,Thi5,D-Tic7,Oic8]BK, [D-Arg0,Hyp3,Thi5,D-Phe7,Thi8]BK, [D-Arg0,Hyp3,Igl5,D-Phe(5F)7,Oic8]BK, and [D-Arg0,Hyp3,Igl5,D-Igl7,Oic8]BK. The peptides were immobilized on a sensor surface consisting of silver (AgNPs) and gold (AuNPs) nanoparticles. These sensors have uniform particle sizes and small size distributions. Thanks to fast synthesis, easy handling, and reproducible results, these sensors enable routine testing. The vibrational structure of these peptides could not be determined using classical vibrational methods (Raman and IR) or surface-enhanced methods (SERS and SEIRA). This work presents the results of that research. Additionally, the SEIRA spectrum for BK or its analogs has not yet been published. This study presents research using SERS and SEIRA that shows that AgNP and AuNP sensors can detect the peptides under investigation. SERS is a more selective method than SEIRA because it allows for the differentiation of peptides based on the enhancement of certain bands in the SERS spectra. Furthermore, each peptide uniquely interacts with AuNPs, whereas all peptides bind to AgNPs via the C-terminus in different orientations. Consequently, the AuNP sensor is more selective than the AgNP sensor. Some bands were selected as markers for the sensing of specific peptides.

Structure and interactions of the proteins from the contact system.

IntroductionThis study aimed to investigate the acute effects of statins on maternal and fetoplacental vascular reactivity in vessels from pregnancies affected by pre-eclampsia (PE), a leading cause of maternal and fetal morbidity and mortality. Statins have been proposed as a candidate therapy due to their pleiotropic effects but evidence of statins’ ability to ameliorate the observed endothelial dysfunction in PE is lacking.MethodsHuman chorionic plate arteries (CPAs) and omental arteries (OAs) from normal and PE pregnancies were mounted on a wire myograph. Contraction was assessed with KPSS and the thromboxane mimetic U46619. Arteries were incubated for 2 h with 1 µM or 10 µM pravastatin, pitavastatin or simvastatin (pitavastatin only in OAs). U46619 dose–response curves were repeated or dose-response curves with NO-donor SNP or endothelium-dependent bradykinin (BK) performed following U46619 pre-constriction.ResultsCPAs from normal and PE pregnancies showed similar responses following exposure to the vasoconstrictive agent U46619 and the relaxatory agent SNP. Short-term exposure to pravastatin, simvastatin and pitavastatin did not cause detrimental effects on CPA reactivity. Acute exposure of OAs from PE pregnancies to pitavastatin (1 µM) did not reduce U46619-mediated contraction or enhance BK-mediated relaxation of vessels although in this study ex vivo endothelial function of OAs from PE pregnancies was not different to those in normotensive pregnancy pre incubation.DiscussionIn conclusion, this study did not demonstrate an effect on vascular reactivity of maternal systemic or fetoplacental arteries following acute treatment of statins. Future studies investigating the effect of longer-term statin exposure on maternal and fetoplacental vascular reactivity may help towards treatment strategies for vascular dysfunction in PE-affected patients.

Endothelial dysfunction plays a central role in COVID-19 pathogenesis, by affecting vascular homeostasis and worsening thromboinflammation. This imbalance may contribute to blood–brain barrier (BBB) disruption, which has been reported in long COVID-19 patients with neurological sequelae. The kallikrein–kinin system (KKS) generates bradykinin (BK), a proinflammatory peptide that induces microvascular leakage via B2R. Under inflammatory conditions, BK is converted to Des-Arg-BK (DABK), which activates B1R, a receptor upregulated in inflamed tissues. DABK is degraded by ACE2, the main SARS-CoV-2 receptor; thus, viral binding and ACE2 downregulation may lead to DABK/B1R imbalance. Here, we investigated these interactions using human brain microvascular endothelial cells (HBMECs), as a model of the BBB. Since endothelial cell lines express low levels of ACE2, HBMECs were modified with an ACE2-carrying pseudovirus. SARS-CoV-2 replication was confirmed by RNA, protein expression, and infectious particles release. Infection upregulated cytokines and endothelial permeability, enhancing viral and leukocyte transmigration. Additionally, viral replication impaired ACE2 function in HBMECs, amplifying the response to DABK, increasing nitric oxide (NO) production, and further disrupting endothelial integrity. Our findings reveal a mechanism by which SARS-CoV-2 impacts the BBB and highlights the ACE2/KKS/B1R axis as a potential contributor to long COVID-19 neurological symptoms.

This study aimed to evaluate the effects of selected phosphodiesterase-4 inhibitors (PDE-4 inhibitors)-roflumilast, ibudilast, and crisaborole-on the activity of blood coagulation factor XII (FXII). In the intrinsic coagulation pathway, FXII is known to initiate the kallikrein-kinin system (KKS), causing an increase in the system expression, which ultimately leads to inflammation and coagulation states. Additionally, the activation of KKS downstream effectors leads to inflammation. Inflammation signaling was found to be initiated when the bradykinin (BK) protein binds to its B2 receptor because of the FXII-dependent pathway activation. BK abnormalities can cause a critical condition, hereditary angioedema (HAE), which is characterized by recurring serious swelling. While it is considered unnecessary for hemostasis, FXII is an important enzyme for pathogenic thrombosis. Because of this special characteristic, FXII is a desirable therapeutic target. Our hypothesis is to identify the inhibitory effects of roflumilast, ibudilast, and crisaborole on the activated FXII and to reveal their beneficial impacts in the reduction of the pathogenesis of FXII-related conditions, HAE, and thrombosis. In a current study, we presented the inhibitory effect of tested drugs on the main target activated factor XII (FXIIa) as well as two other plasma protease enzymes included in the target pathway, plasma kallikrein and FXIa. To achieve our aim, in vitro chromogenic enzymatic assays were utilized to assess the inhibitory effects of these drugs by monitoring the amount of para-nitroaniline (pNA) chromophore released from the substrate of FXIIa, FXIa, or plasma kallikrein. Our study findings exhibited that among assessed PDE-4 inhibitor drugs, roflumilast at micromolar concentrations significantly inhibited FXIIa in a dose-dependent manner. The FXIIa was clearly suppressed by roflumilast, but not the other related KKS members, plasma kallikrein, or the activated factor XI. On the other hand, ibudilast and crisaborole showed no inhibitory effects on the activities of all enzymes. Overall, roflumilast could be used as a lead compound for developing a novel multifunctional therapeutic drug used for the prevention of HAE or thrombotic disorders.

Cardiogenic dementia is associated with an increased risk of mortality in heart failure patients. Cognitive decline is more prevalent in females compared to males, of which cardiac function is the strongest predictor. We have previously demonstrated cerebrovascular insufficiency in female Ossabaw swine with cardiometabolic heart failure (CM-HF), and further shown improved cardiac function and ventricular-vascular interactions by a novel RSK3/mAKAPβ targeted gene therapy. The RSK3/mAKAPβ anchoring disruptor peptide (RBD) displaces RSK3 on mAKAPβ by adeno-associated virus (AAV)-based expression of RBD (AAV9sc.RBD). The goal of this study was to examine the effects of RBD gene therapy on transcriptomic signaling in both coronary and cerebral arterioles in an Ossabaw swine model of CM-HF. Female Ossabaw pigs were assigned to HF control (HF; n=4-5) and HF AAV9sc.RBD-treated (HF+RBD; n=4-7). Animals were fed a Western diet (3 mo. old) and aortic banded (6 mo. old) prior to terminal experiments (12 mo. old). Peripheral intravenous saline containing 3 x 10 14 viral genomes (vg) of AAV9sc.RBD biologic was infused immediately after aortic banding. Wet lung weight was decreased in HF+RBD animals compared to HF (283±19 vs. 423±64 g; unpaired t-test, p&lt;0.05) and was associated with improved diastolic function evident by a decrease in the end diastolic pressure-volume relationship (EDPVR: 0.039±0.004 vs. 0.022±0.003 mmHg/mL; unpaired t-test, p&lt;0.05). RBD also increased stroke volume (40±2 vs. 50±2 mL; unpaired t-test, p&lt;0.05) and improved hemodynamic coupling between the left ventricle and peripheral vasculature. RBD increased endothelial-dependent dilation to bradykinin (BK) compared to HF (Two-way ANOVA, Group x Dose interaction; p&lt;0.05) in isolated second order (2A) pial arterioles, which was unaltered in the presence of L-NAME (nitric oxide synthase inhibitor) and indomethacin (prostaglandin synthesis inhibitor). RNA sequencing identified 26 differentially expressed genes (DEGs) in cerebral arterioles and 11 DEGs in coronary arterioles. Ingenuity pathway analysis (IPA) on cerebral arterioles predicted inhibition of upstream regulator POR (z-score &gt; -2), a gene associated with decreased fibrosis and organismal death, as well as increased sterol synthesis and quantity of leukocytes. Whereas gene enrichment of the coronary arterioles predicted cytokine (TNF, z-score=1.96, and IL1B, z-score=1.98) and growth factor activation (TGFB1, z-score=1.99). These findings demonstrate RBD improvement in coronary and cerebral vascular function occurs through distinct transcriptomic adaptations involving pathways associated with fibrosis, leukocyte recruitment, cytokine production, and growth factor activation. MU Tier 1 Sequencing Grant and Department of Defense (DOD) Grant W81XWH-18-1-0179 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Epileptic seizures cause brain oxidative stress which contributes to long-term cerebral vascular dysfunction and blood-brain barrier (BBB) damage leading to cerebrovascular disease and potential neurological deficits. Seeking new therapeutic interventions to reduce brain oxidative stress is crucial for preventing seizure-induced cerebrovascular and neurological complications. NADPH oxidase (NOX1-NOX5 isoforms) is a potent source of reactive oxygen species (ROS) in the brain. We tested the effects of setanaxib (GKT831), a first class selective inhibitor of NADPH oxidase isoforms NOX1/NOX4, on ROS generation in the neurovascular unit and on the cerebrovascular outcome of epileptic seizures in newborn pigs. Setanaxib (5-10 µM) exhibited strong antioxidant and cytoprotective effects in primary cerebral endothelial cells and astrocytes exposed to seizure-related excitatory and inflammatory pro-oxidants glutamate and TNF-alpha. Glutamatergic epileptic seizures were induced by bicuculline, and postictal cerebral vascular function and BBB permeability were evaluated 48 h after seizures in saline-control and setanaxib-treated pigs. Setanaxib (5 mg/kg i.p.) was administered 30 min before seizures (preventive protocol) or 10 min after bicuculline (therapeutic protocol). Postictal cerebral vascular function was evaluated 48 h after seizures by the responses of pial arterioles to endothelium- and astrocyte-dependent vasodilators bradykinin (BK), ADP, glutamate (Glu) and the glutamate receptor agonists NMDA and quisqualic acid (Quis). In the saline control post-epileptic pigs, the responses to all endothelium- and astrocyte-dependent dilators were greatly reduced compared to intact pigs. In contrast, in newborn pigs treated by setanaxib either before or during seizures, all postictal cerebral vascular responses were largely preserved. Furthermore, setanaxib administered preventively or therapeutically fully prevented BBB leakage detected by Evans Blue extravasation 48 h after seizures. In summary, setanaxib is a promising therapeutic agent to reduce brain oxidative stress and to prevent cerebrovascular disease and BBB disruption caused by epileptic seizures. Grant Support: R01NS105655, R01NS134659 (Parfenova) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract: Inflammation is a defense response of the body that occurs due to injury or bacterial infection, allergens, autoimmune disease, etc. PGs (Prostaglandins), leukotrienes, histamine, BK (bradykinin), and ILs (interleukins) are the main chemical mediators involved in inflammation and its associated diseases. The major pathway is the formation of arachidonic acid connected with the cyclooxygenase (COX) enzyme and 5-lipoxygenase enzyme in the synthesis of prostaglandins and leukotrienes, respectively. Anti-inflammatory agents primarily target cyclooxygenases (COX 1 and 2), phospholipase A2 (PLA2), and lipoxygenase (LOX). Some major patents are covered in this review article. This review also covers the journey of anti-inflammatory activities of tetrahydrocarbazole structure-based derivatives from 1976 till date. Researchers around the world have designed and synthesized novel tetrahydrocarbazoles based on the indomethacin structure under the NSAIDs category using Structure-based Drug Design (SBBD), which have displayed significant in vitro and in vivo anti-inflammatory activities. Tetrahydrocarbazole structures linked with other potential pharmacophores, including chalconyl, isoxazolinyl, pyrazolinyl, and diazoles, have also been evaluated for anti-inflammatory activities. Major substitutions, either halogen or electron-releasing groups on tetrahydrocarbazole structure, play a crucial role in its anti-inflammatory activities. Moreover. reported N-9 substituted tetrahydrocarbazoles have been observed to display significant anti-inflammatory activities. These THCz derivatives target the COX-2 enzyme, which plays a crucial role in inflammation and also in cancer. Lastly, this review encourages scientists to design and synthesize novel compounds based on tetrahydrocarbazole structure to address inflammation and its associated diseases.

Remote ischemic postconditioning (RIPostC) is known to improve motor function recovery in animal models, but its efficacy in alleviating cognitive impairment caused by ischemic stroke remains unclear. This study aims to investigate the beneficial role of RIPostC in recovering cognitive impairment induced by cerebral ischemia-reperfusion injury (CIRI). Building upon our previous research findings, we proved that the TK/BK/B2R pathway is crucial for understanding the crosstalk between cognitive impairment and RIPostC. Additionally, in vitro experiments were conducted using the oxygen glucose deprivation/re-oxygenation (OGD/r) HT-22 cell model, which revealed that the mechanism by which RIPostC suppressed mitochondrial apoptosis was mainly through the activation of the B2R/PI3K/AKT signaling pathway, thereby protecting neurons in the ischemic hippocampus from ischemic damage. To investigate the effect of RIPostC on cognitive function recovery following ischemic stroke, we established a rat model using left middle cerebral artery occlusion reperfusion (MCAO/r). 48h after MCAO/r, rats were subjected to 3 circles of RIPostC therapy daily for 12 consecutive days. HOE140 was used to antagonize the bradykinin 2 receptor (B2R). Cognitive function was assessed using a modified neurological severity score, the Morris water maze, and the novel object recognition test. Local infarct volume in the hippocampus was measured through MRI scanning. The apoptosis rate of hippocampal neurons was quantified using TUNEL staining. Protein expression levels of kallikrein (TK) and mitochondrial apoptosis-related proteins, Cyt c, Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-9, were detected in ischemic hippocampal tissue using Western blot (WB). The expression of bradykinin (BK) in serum and the ischemic penumbra was measured using an enzyme-linked immunosorbent (ELISA) assay. In the cell experiments, the HT-22 cell line and OGD/r model were used to simulate in vitro hippocampal ischemia. WB was performed to detect the expression of apoptosis-related proteins and PI3K/AKT pathway proteins. The apoptosis rate of HT-22 cells was detected using Annexin-V/PI flow cytometry and a cell viability kit. JC-1 staining and reactive oxygen species staining were used to evaluate mitochondrial condition. The PI3K/AKT pathway was inhibited using LY294002. RIPostC significantly upregulated the concentrations of TK and BK in the ischemic hippocampus. Behavioral function tests demonstrated that daily RIPostC therapy for 12days significantly promoted cognitive function recovery in MCAO/r rats. Through MRI analysis, we found that RIPostC therapy effectively reduced the infarct volume in the hippocampus. Additionally, TUNEL staining and WB results of apoptosis-related proteins showed that RIPostC therapy significantly reduced apoptosis of hippocampal neurons. However, the therapeutic effect of RIPostC was reversed by the B2R antagonist HOE14, indicating that the TK/BK/B2R pathway mediated the neuroprotective effect of RIPostC. Cell experiments further confirmed that BK/B2R significantly attenuated mitochondrial apoptosis induced by ischemia-hypoxia injury in HT-22 cells. In vivo and in vitro results from WB demonstrated that the BK/B2R pathway activated the PI3K/AKT signaling pathway. Finally, the PI3K inhibitor LY294002 reversed the anti-apoptotic effect induced by BK/B2R. RIPostC therapy effectively inhibited mitochondrial apoptosis of hippocampal neurons and significantly alleviated cognitive dysfunction associated with CIRI by regulating the TK/BK/B2R-medated PI3K/AKT pathway. In conclusion, RIPostC represents a promising therapeutic strategy for combating cognitive dysfunction by inhibiting cell apoptosis in hippocampus. Moreover, our results suggest that RIPostC may have a broader protective effect against apoptosis in other ischemia-reperfusion-related diseases.

External factors in ulcerative colitis (UC) exacerbate colonic epithelial permeability and inflammatory responses. Keratin 1 (KRT1) is crucial in regulating these alterations, but its specific role in the progression of UC remains to be fully elucidated. To explore the role and mechanisms of KRT1 in the regulation of colonic epithelial permeability and inflammation in UC. A KRT1 antibody concentration gradient test, along with a dextran sulfate sodium (DSS)-induced animal model, was implemented to investigate the role of KRT1 in modulating the activation of the kallikrein kinin system (KKS) and the cleavage of bradykinin (BK)/high molecular weight kininogen (HK) in UC. Treatment with KRT1 antibody in Caco-2 cells suppressed cell proliferation, induced apoptosis, reduced HK expression, and increased BK expression. It further downregulated intestinal barrier proteins, including occludin, zonula occludens-1, and claudin, and negatively impacted the coagulation factor XII. These changes led to enhanced activation of BK and HK cleavage, thereby intensifying KKS-mediated inflammation in UC. In the DSS-induced mouse model, administration of KRT1 antibody mitigated colonic injury, increased colon length, alleviated weight loss, and suppressed inflammatory cytokines such as interleukin (IL)-1, IL-6, tumor necrosis factor-α. It also facilitated repair of the intestinal barrier, reducing DSS-induced injury. KRT1 inhibits BK expression, suppresses inflammatory cytokines, and enhances markers of intestinal barrier function, thus ameliorating colonic damage and maintaining barrier integrity. KRT1 is a viable therapeutic target for UC.

Bradykinin attenuates NiSO4-induced autophagy in MIN6 cells and protects islet function in mice by regulating the PI3K/AKT/mTOR signaling pathway.