H2 Relaxin Research Articles

H3 relaxin ameliorates mitochondrial quality control and apoptosis in cardiomyocytes of type 2 diabetic rats via activation of the AMPK pathway

PurposeThis study aims to explore the protective mechanism of H3 relaxin treatment for type 2 diabetic cardiomyopathy, focusing on its effects on the mitochondrial quality control system and cardiomyocyte apoptosis. MethodsA type 2 diabetes rat model was induced with a high-fat diet and streptozotocin. The treatment group received H3 relaxin for 2 weeks. In cellular studies, H9C2 cardiomyocytes were treated with high glucose and palmitic acid, followed by H3 relaxin, MFN2 lentivirus, and AMPK inhibitor compound C. Mitochondrial quality control, membrane potential, ROS, and apoptosis markers were assessed. ResultsH3 relaxin improved apoptosis markers, reduced mitochondrial fission (Drp1, Fis1) and mitophagy proteins (parkin, PINK1), and increased mitochondrial fusion proteins (MFN2, OPA1). It restored mitochondrial membrane potential, lowered ROS, and showed anti-apoptotic effects dependent on MFN2. AMPK pathway activation by H3 relaxin was crucial, as compound C largely negated these benefits. ConclusionH3 relaxin improves the mitochondrial quality control system and reduces high glucose and palmitic acid-induced apoptosis by upregulating MFN2. This protective effect is achieved through the activation of the AMPK pathway.

Abstract 16: A Novel H2 Relaxin B-Chain-Only Peptide Variant B7-33 Improves The Pathophysiology Of Placental Ischemia In The Reduced Uterine Perfusion Pressure Rat Model Of Preeclampsia

Background: Preeclampsia (PE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. The literature suggests the potential therapeutic role of H2 relaxin in PE. A novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. Objective: The goal of this study was to test the hypothesis that a novel H2 relaxin B-chain-only peptide variant B7-33 could improve the pathophysiology of placental ischemia in the Reduced Uterine Perfusion Pressure (RUPP) rat model of PE. Methods: The efficacy of B7-33 was evaluated in the RUPP model as described previously. RUPP rats are randomly assigned to 4 groups (N=8/group): 1) vehicle, 2) B733; 3) B733-Fc; and 4) B733-HSA. Rats are dosed twice weekly (i.v.) from GD10 to GD 20. On GD 18, rats are anesthetized with isoflurane, and carotid arterial catheters are inserted into the carotid artery, tunneled under the skin, and externalized at the back of the neck. On the following day, mean arterial pressure (MAP) is monitored with a pressure transducer (Cobe III Tranducer CDX Sema) and recorded continuously for 30 min. On GD18, uterine artery resistance index (UARI) of rats is measured by Doppler sonography. The nitric oxide bioavailability, soluble fms-like tyrosine kinase-1 (sFlt-1), and TNF-α) were measured by commercially available kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: The RUPP rats have increased MAP (122.2 ± 8.1 mm Hg), plasma TNF-α (223 ±11.4 pg/mL), and plasma sFlt-1(863 ±18.2 pg/mL) along with decreased NO index (14±2 µM) compared to normal pregnancy: MAP (102 ± 5.2 mm Hg); plasma TNF-α (28 ± 4.1 pg/mL); plasma sFlt-1(244 ± 9.4 pg/mL) and NO index (26 ±4.1 µM). Treatment with B733 and B7-33 fusion proteins significantly (*p<0.05) lowers MAP, plasma TNF-α, and plasma sFlt-1 and increased NO index back to that of normal pregnancy. The B7-33 data are consistent with earlier data with serelaxin in the RUPP model. All fusions tested ameliorate hypertension in the RUPP animals. B7-33 normalizes BP and proteinuria in the DOCA rat model of preE. Conclusion: The conclusion of the study is that both B7-33 and B7-33 fusion proteins demonstrate efficacy in attenuating the symptoms of PE including hypertension and inflammation in RUPP model. We conclude that B7-33 is an ideal candidate for development as a novel therapeutic in preE.

Seleno-relaxin analogues: effect of internal and external diselenide bonds on the foldability and a fibrosis-related factor of endometriotic stromal cells.

Human relaxin-2 (H2 relaxin) is a peptide hormone of about 6 kDa, first identified as a reproductive hormone involved in vasoregulation during pregnancy. It has recently attracted strong interest because of its diverse functions, including anti-inflammatory, anti-fibrotic, and vasodilatory, and has been suggested as a potential peptide-based drug candidate for a variety of diseases. Mature H2 relaxin is constituted by the A- and B-chains stabilized by two interchain disulfide (SS) bridges and one intrachain SS linkage. In this study, seleno-relaxins, SeRlx-α and SeRlx-β, which are [C11UA,C11UB] and [C10UA,C15UA] variants of H2 relaxin, respectively, were synthesized via a one-pot oxidative chain assembly (folding) from the component A- and B-chains. The substitution of SS bonds in a protein with their analogue, diselenide (SeSe) bonds, has been shown to alter the physical, chemical, and physiological properties of the protein. The surface SeSe bond (U11A-U11B) enhanced the yield of chain assembly while the internal SeSe bond (U10A-U15A) improved the reaction rate of the folding, indicating that these bridges play a major role in controlling the thermodynamics and kinetics, respectively, of the folding mechanism. Furthermore, SeRlx-α and SeRlx-β effectively reduced the expression of a tissue fibrosis-related factor in human endometriotic stromal cells. Thus, the findings of this study indicate that the S-to-Se substitution strategy not only enhances the foldability of relaxin, but also provides new guidance for the development of novel relaxin formulations for endometriosis treatment.

Abstract P426: Novel Peptide B7-33 Demonstrates Efficacy In Rat Models Of Preeclampsia

Background: Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. The literature suggests potential therapeutic role of H2 relaxin in PreE. Due to the complex insulin-like structure of relaxin (A- and B- chains, 53 amino acids, 3 disulfide bonds), a novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. Objective: This single-chain peptide displayed equivalent efficacy to the natural H2 relaxin in several functional assays both in vitro and in vivo . The Aim of this study is to evaluate whether B7-33 attenuates preE syndrome in rat models of PreE. Methods: Rat Model 1. The efficacy of B7-33 was evaluated in the Reduction of Uterine Perfusion Pressure (RUPP) model as described previously. RUPP rats are randomly assigned to 4 groups (N=8/group): 1) vehicle, 2) B733; 3) B733-Fc; and 4) B733-HSA. Rats are dosed twice weekly (i.v.) from GD10 to GD 20. Rat Model 2. Timed-pregnant rats are used, and the model is conducted as previously described. DOCA and saline administration begin at approximately GD2. Three groups of animals are studied (n=8 per group). 1) normal pregnant rats; 2) pregnant animals injected (i.p.) initially with 12.5 mg of DOCA, followed by a weekly injection of 6.5 mg, and whose drinking water was replaced with 0.9% saline; 3) rats administered DOCA and saline as for Group 2, and given B733 35 ug i.p. biweekly from GD10-20. BP, proteinuria and inflammatory markers were evaluated. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: RUPP rats have increased MAP, plasma TNF-α, and plasma sFlt-1 along with decreased NO index compared to normal pregnancy. Treatment with B733 and B7-33 fusion proteins lowers MAP, TNF-α, and sFlt-1 back to that of normal pregnancy. B7-33 data are consistent with earlier data with serelaxin in the RUPP model. All fusions tested ameliorate hypertension in the RUPP animals. B7-33 normalizes BP and proteinuria in the DOCA rat model of preE. Conclusion: Both B7-33 and B7-33 fusion proteins attenuate preE syndrome in RUPP and DOCA rat models of PreE. We conclude that B7-33 is an ideal candidate for development as a novel therapeutic in preE.

Synthesis of A11Cys-B11Cys Disulfide Surrogates of H2 Relaxin through an Intermolecular Native Chemical Ligation-Assisted Diaminodiacid Strategy.

We report an intermolecular native chemical ligation-assisted diaminodiacid strategy for the flexible construction of A11Cys-B11Cys disulfide surrogates of H2 relaxin. The practicality of this strategy was evidenced by the synthesis of four new H2 relaxin analogs, among which H2-2a-B28Ile is found to exhibit improved potency, selectivity, and stability compared with native H2 relaxin.

Further Developments towards a Minimal Potent Derivative of Human Relaxin-2.

Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.

Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

Development of Novel High-Affinity Antagonists for the Relaxin Family Peptide Receptor 1.

H2 relaxin is a peptide hormone that exerts its biological actions through the G protein-coupled receptor, RXFP1. The numerous important biological functions of H2 relaxin, including potent renal, vasodilatory, cardioprotective, and anti-fibrotic actions, have resulted in considerable interest in its use as a therapeutic for various cardiovascular diseases and other fibrotic indications. Interestingly though, H2 relaxin and RXFP1 have been shown to be overexpressed in prostate cancer, allowing for the downregulation or blocking of relaxin/RXFP1 to decrease prostate tumor growth. These findings suggest the application of an RXFP1 antagonist for the treatment of prostate cancer. However, these therapeutically relevant actions are still poorly understood and have been hindered by the lack of a high-affinity antagonist. In this study, we chemically synthesized three novel H2 relaxin analogues that have complex insulin-like structures with two chains (A and B) and three disulfide bridges. We report here the structure-activity relationship studies on H2 relaxin that resulted in the development of a novel high-affinity RXFP1 antagonist, H2 B-R13HR (∼40 nM), that has only one extra methylene group in the side chain of arginine 13 in the B-chain (ArgB13) of H2 relaxin. Most notably, the synthetic peptide was shown to be active in a mouse model of prostate tumor growth in vivo where it inhibited relaxin-mediated tumor growth. Our compound H2 B-R13HR will be an important research tool to understand relaxin actions through RXFP1 and may be a potential lead compound for the treatment of prostate cancer.

A Lipidated Single-B-Chain Derivative of Relaxin Exhibits Improved In Vitro Serum Stability without Altering Activity.

Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin's receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.

Single-Shot Solid-Phase Synthesis of Full-Length H2 Relaxin Disulfide Surrogates.

Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next-generation drugs. Separate synthesis of multiple peptide fragments and tedious chain-to-chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre-made diaminodiacid bridge at A-B chain terminal disulfide can be easily and rapidly synthesized by a single-shot automated solid-phase synthesis and expedient one-step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.

Single‐Shot Solid‐Phase Synthesis of Full‐Length H2 Relaxin Disulfide Surrogates

AbstractChemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next‐generation drugs. Separate synthesis of multiple peptide fragments and tedious chain‐to‐chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre‐made diaminodiacid bridge at A‐B chain terminal disulfide can be easily and rapidly synthesized by a single‐shot automated solid‐phase synthesis and expedient one‐step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.

Porcine Relaxin but Not Serelaxin Shows Residual Bioactivity after In Vitro Simulated Intestinal Digestion-Clues for the Development of New Relaxin Peptide Agonists Suitable for Oral Delivery.

Despite human recombinant H2 relaxin or serelaxin holding promise as a cardiovascular drug, its actual efficacy in chronic treatment of heart failure patients was hampered by the need to be administered by multiple daily IV injections for a long time, with obvious drawbacks in terms of patients' compliance. This in vitro study aimed at exploring the molecular background for a possible administration of the peptide hormone relaxin by the oral route. Serelaxin and purified porcine relaxin (pRLX) were subjected to simulated intestinal fluid (SIF) enzymatic digestion in vitro to mimic the behavior of gastroprotective formulations. The digestion time course was studied by HPLC, and the relative bio-potency of the intact molecules and their proteolytic fragments was assessed by second messenger (cAMP) response in RXFP1 relaxin receptor-bearing THP-1 human monocytic cells. Both intact proteins (100 ng/mL) induced a significant cAMP rise in THP-1 cells. Conversely, SIF-treated serelaxin showed a brisk (30 s) bioactivity decay, dropping down to the levels of the unstimulated controls at 120 s, whereas SIF-treated pRLX retained significant bioactivity for up to 120 s. After that, it progressively declined to the levels of the unstimulated controls. HPLC analysis indicates that this bioactivity could be ascribed to a minor component of the pRLX sample more resistant to proteolysis. When identified and better characterized, this peptide could be exploited for the development of synthetic relaxin agonists suitable for oral formulations.

Post-stroke administration of H2 relaxin reduces functional deficits, neuronal apoptosis and immune cell infiltration into the mouse brain

Brain inflammation and apoptosis contribute to neuronal damage and loss following ischaemic stroke, leading to cognitive and functional disability. It is well-documented that the human gene-2 (H2)-relaxin hormone exhibits pleiotropic properties via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), including anti-inflammatory and anti-apoptotic effects, thus making it a potential therapeutic for stroke. Hence, the current study investigated whether post-stroke H2-relaxin administration could improve functional and histological outcomes. 8–12-week-old male C57BL/6 mice were subjected to sham operation or photothrombotic stroke and intravenously-administered with either saline (vehicle) or 0.02, 0.2 or 2 mg/kg doses of recombinant H2-relaxin at 6, 24 and 48 h post-stroke. Motor function was assessed using the hanging wire and cylinder test pre-surgery, and at 24 and 72 h post-stroke. Brains were removed after 72 h and infarct volume was assessed via thionin staining, and RXFP1 expression, leukocyte infiltration and apoptosis were determined by immunofluorescence. RXFP1 was identified on neurons, astrocytes and macrophages, and increased post-stroke. Whilst H2-relaxin did not alter infarct volume, it did cause a dose-dependent improvement in motor function at 24 and 72 h post-stroke. Moreover, 2 mg/kg H2-relaxin significantly decreased the number of apoptotic cells as well as macrophages and neutrophils within the ischaemic hemisphere, but did not alter T or B cells numbers. The anti-inflammatory and anti-apoptotic effects of H2-relaxin when administered at 6 h post-cerebral ischaemia may provide a novel therapeutic option for patients following ischaemic stroke.

Post-Stroke Administration of H2 Relaxin Reduces Functional Deficits, Neuronal Apoptosis and Immune Cell Infiltration into the Mouse Brain

Post-Stroke Administration of H2 Relaxin Reduces Functional Deficits, Neuronal Apoptosis and Immune Cell Infiltration into the Mouse Brain

Changes in microRNA Expression Profiles in Diabetic Cardiomyopathy Rats Following H3 Relaxin Treatment.

MicroRNAs (miRNAs) are noncoding RNAs that play an important role in the mechanisms of diabetic cardiomyopathy (DCM); however, whether human recombinant relaxin-3 (H3 relaxin) inhibits myocardial injury in DCM rats and the underlying mechanisms involving miRNAs remain unknown. miRNA expression profiles were detected using miRNA microarray and bioinformatics analyses of myocardial tissues from control, DCM, and H3 relaxin-administered DCM groups, and the regulatory mechanisms of the miRNAs were investigated. A total of 5 miRNAs were downregulated in the myocardial tissues of DCM rats and upregulated in H3 relaxin-treated DCM rats, and 1 miRNA (miRNA let-7d-3p) was increased in the myocardial tissue of DCM rats and decreased in H3 relaxin-treated DCM rats as revealed by miRNA microarray and validated by real-time polymerase chain reaction. Important signaling pathways were found to be triggered by the differentially expressed miRNAs, including metabolism, cancer, Rap1, PI3K-Akt, and MAPK signaling pathways. The study revealed that H3 relaxin improved glucose uptake in DCM rats, potentially via the regulation of miRNA let-7d-3p.

Inhaled B7 alleviates bleomycin-induced pulmonary fibrosis in mice

Treatment options for the progression of pulmonary fibrosis (PF), which ultimately causes respiratory failure, are limited. According to recent studies, recombinant human relaxin is potentially therapeutic against fibrosis and contraction during pulmonary damage. However, the production of recombinant H2 relaxin is laborious and expensive, limiting its extensive application. Thankfully, alternative research has revealed that treatment with a single-chain peptide of relaxin attenuates organ fibrosis in rodent models too, with the production of a single-chain peptide of relaxin simple and cheap; it could be therapeutic against idiopathic pulmonary fibrosis. Here, we explored the probable inhibiting effects of B7, a B chain of recombinant human relaxin, on bleomycin-induced pulmonary inflammation. Inhaled B7 efficiently reduced the number of inflammatory leukocytes and neutrophils in the bronchoalveolar lavage fluid of mice with bleomycin-induced PF, significantly improved the structure of the damaged alveolar, reduced collagen deposition, suppressed the main pathological features of idiopathic pulmonary fibrosis, i.e. the expression of both pulmonary α-smooth muscle actin and pulmonary vimentin, and inhibited the transcription of inflammation and collagen deposition-related mRNAs, including fibronectin, α-smooth muscle actin (α-SMA), interleukin-1β (IL-1β), interleukin-6 (IL-6), and alpha-1 type 1 collagen (Col-1a), and the expression of inflammation-related proteins, such as IL-1β, IL-6, chemokines (KC), TIMP metallopeptidase inhibitor 1 (TIMP-1), and hydroxyproline (Hyp). Overall, our findings suggest that inhaled B7 exerts beneficial effects against pulmonary fibrosis via attenuating inflammation. It could be developed into a simple, highly effective therapeutic approach for pulmonary fibrosis.

Structural Insights into the Unique Modes of Relaxin-Binding and Tethered-Agonist Mediated Activation of RXFP1 and RXFP2

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors.

Relaxin peptides reduce cellular damage in cultured brain slices exposed to transient oxygen–glucose deprivation: an effect mediated by nitric oxide

The effect of treatment with human relaxins on cell death was studied in oxygen- and glucose-deprived brain slices. In addition, involvement of nitric oxide and the relaxin receptor, RXFP3, was studied. Brain slices ( n = 12–18/group) were cultured under standard conditions for two weeks and then exposed to: ( i) an oxygenated balanced salt solution, ( ii) a deoxygenated, glucose-free balanced salt solution (OGD media), or ( iii) OGD media containing 10−7mol/L H2 relaxin, 10−7mol/L H2 relaxin with 50 μmol/L L-NIL, 10−7mol/L H3 relaxin, or 10−7mol/L H3 relaxin with 50 μmol/L L-NIL. Cell death was assessed using propidium iodide fluorescence. In a separate experiment, 10−5mol/L R3 B1-22R (an antagonist of RXFP3) was added to both H2 and H3 relaxin treatments. H2 and H3 relaxin treatment reduced cell damage or death in OGD slices and L-NIL partially attenuated the effect of H3 relaxin. Antagonism of RXFP3 blocked the effect of H3 but not H2 relaxin. These data increase our understanding of the role of relaxin ligands and their receptors in protecting tissues throughout the body from ischemia and reperfusion injury.

H3 Relaxin Alleviates Migration, Apoptosis and Pyroptosis Through P2X7R-Mediated Nucleotide Binding Oligomerization Domain-Like Receptor Protein 3 Inflammasome Activation in Retinopathy Induced by Hyperglycemia.

Introduction: P2X7R excitation-interrelated NLRP3 inflammasome activation induced by high glucose contributes to the pathogenesis of diabetic retinopathy (DR). Relaxin-3 is a bioactive peptide with a structure similar to insulin, which has been reported to be effective in diabetic cardiomyopathy models in vivo and in vitro. However, it is not known whether relaxin-3 has a beneficial impact on DR, and the underlying mechanisms of the effect are also remain unknown. Methods and Results: The retinas of male streptozotocin (STZ)-induced diabetic Sprague-Dawley (SD) rats were characterized. Human retinal microvascular endothelial cells (HRMECs) were used to evaluate the anti-inflammatory, antiapoptotic, antipyroptotic and anti-migration effects of H3 relaxin by transmission electron microscopy, wound-healing assay, transwell assay, flow cytometry, cytokine assays and western-blot analysis. After H3 relaxin treatment, changes of the ultrastructure and expression of NLRP3 inflammasome related proteins in the retinas of rats were compared with those in the diabetic group. In vitro, H3 relaxin played a beneficial role that decreased cell inflammation, apoptosis, pyroptosis and migration stimulated by advanced glycation end products (AGEs). Moreover, inhibition of P2X7R and NLRP3 inflammasome activation decreased NLRP3 inflammasome-mediated injury that similar to the effects of H3 relaxin. H3 relaxin suppressed the stimulation of apoptosis, pyroptosis and migration of HRMECs in response to AGEs mediated by P2X7R activation of the NLRP3 inflammasome. Conclusion: Our findings provide new insights into the mechanisms of the inhibitory effect of H3 relaxin on AGE-induced retinal injury, including migration, apoptosis and pyroptosis, mediated by P2X7R-dependent activation of the NLRP3 inflammasome in HRMECs.

H3 relaxin protects against calcium oxalate crystal-induced renal inflammatory pyroptosis.

ObjectivesCalcium oxalate (CaOx) crystals can activate inflammatory cytokines by triggering inflammasomes, which cause damage to the adhered epithelium, a dysfunctional microenvironment and even renal failure. However, a comprehensive and in‐depth understanding of the mechanisms underlying the effects of these crystals on damage and cytokine function in renal tubular epithelial cells (TECs) remains limited and to be explored.Materials and MethodsWe detected the pyroptosis of TECs induced after exposure to CaOx crystals and demonstrated the significance of cytokine activation in the subsequent inflammatory processes through a proteomic study. We then conducted animal and cell experiments to verify relevant mechanisms through morphological, protein, histological and biochemical approaches. Human serum samples were further tested to help explain the pathophysiological mechanism of H3 relaxin.ResultsWe verified that crystal‐induced extracellular adenosine triphosphate (ATP) upregulation via the membrane purinergic 2X7 receptor (P2X7R) promotes ROS generation and thereby activates NLRP3 inflammasome‐mediated interleukin‐1β/18 maturation and gasdermin D cleavage. Human recombinant relaxin‐3 (H3 relaxin) can act on the transmembrane receptor RXFP1 to produce cAMP and subsequently improves crystal‐derived damage via ATP consumption. Additionally, endogenous relaxin‐3 was found to be elevated in patients with renal calculus and can thus serve as a biomarker.ConclusionsOur results provide previously unidentified mechanistic insights into CaOx crystal‐induced inflammatory pyroptotic damage and H3 relaxin‐mediated anti‐inflammatory protection and thus suggest a series of potential therapeutic targets and methods for but not limited to nephrocalcinosis.