Investigation of the protective effect of chitosan against arsenic-induced nephrotoxicity and oxidative damage in rat kidney tissue.

Arsenic is an important metalloid that can cause poisoning in humans and domestic animals. Exposure to arsenic causes cell damage, increasing the production of reactive oxygen species. Chitosan is a biopolymer obtained by deacetylation of chitin with antioxidant and metal ion chelating properties. In this study, the protective effect of chitosan on arsenic-induced nephrotoxicity and oxidative damage was investigated. 32 male Wistar-albino rats were divided into 4 groups of 8 rats each as control group (C), chitosan group (CS group), arsenic group (AS group), and arsenic+chitosan group (AS+CS group). The C group was given distilled water by oral gavage, the AS group was given 100 ppm/day Na-arsenite ad libitum with drinking water, the CS group was given 200 mg/kg/day chitosan dissolved in saline by oral gavage, the AS+CS group was given 100 ppm/day Na-arsenite ad libitum with drinking water and 200 mg/kg/day chitosan dissolved in saline by oral gavage for 30 days. At the end of the 30-day experimental period, 90 mg/kg ketamine was administered intraperitoneally to all rats, and blood samples and kidney tissues were collected. Urea, uric acid, creatinine, P, Mg, K, Ca, Na, Cystatin C (CYS-C), Neutrophil Gelatinase Associated Lipocalin (NGAL) and Kidney Injury Molecule 1 (KIM-1) levels were measured in serum samples. Malondialdehyde (MDA), Glutathione (GSH), Catalase (CAT) and Superoxide dismutase (SOD) levels in the supernatant obtained from kidney tissue were analyzed by ELISA method. Compared with AS group, uric acid and creatinine levels of the AS+CS group were significantly decreased (p<0.001), urea, KIM-1, CYS-C, NGAL, and MDA levels were numerically decreased and CAT, GSH, and SOD levels were numerically increased (p>0.05). In conclusion, based on both biochemical and histopathological-immunohistochemical- immunofluorescence findings, it can be concluded that chitosan attenuates kidney injury and protects the kidney.

Quercetin ameliorates Helicobacter pylori-induced gastric epithelial cell injury by regulating specificity protein 1/lipocalin 2 axis in gastritis.

Helicobacter pylori (HP) infection is the main cause of most cases of gastritis. Quercetin has been shown to have anti-inflammatory, anti-bacterial, and antiviral activities and has been demonstrated to be involved in HP-induced gastric mucosa injury. Moreover, the secretory protein lipocalin-2 (LCN2) was elevated in HP-infected gastric mucosa. Thus, this work aimed to study the interaction between quercetin and LCN2 in HP-triggered gastric injury during gastritis. Human gastric epithelial cell line GES-1 cells were exposed to HP for functional experiments. Cell viability, apoptosis, and inflammation were evaluated by cell counting kit-8, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Levels of genes and proteins were tested using quantitative reverse transcription polymerase chain reaction and western blotting analyses. The interaction between LCN2 and specificity protein 1 (SP1) was validated using chromatin immunoprecipitation assay and dual-luciferase reporter assay. Thereafter, we found quercetin treatment suppressed HP-induced GES-1 cell apoptotic and inflammatory injury and macrophage M1 polarization. LCN2 was highly expressed in HP-infected gastritis patients and HP-infected GES-1 cells, while quercetin reduced LCN2 expression in HP-infected GES-1 cells; moreover, LCN2 knockdown reversed HP-induced GES-1 cell injury and macrophage M1 polarization, and forced expression of LCN2 abolished the protective effects of quercetin on GES-1 cells under HP infection. Mechanistically, SP1 bound to LCN2 promoter and promoted its transcription. Also, SP1 overexpression counteracted the functions of quercetin on HP-stimulated GES-1 cells. In all, quercetin ameliorated HP-induced gastric epithelial cell apoptotic and inflammatory injuries, and macrophage M1 polarization via the SP1/LCN2 axis.

HNRNPA2B1-mediated m6A modification of FOXM1 promotes drug resistance and inhibits ferroptosis in endometrial cancer via regulation of LCN2.

N6-methyladenosine (m6A) methylation is an extensive posttranscriptional RNA modification, and it is associated with various cellular responses, especially in tumor progression. An m6A "reader"-HNRNPA2B1 has been found oncogenic in multiple malignancies. As a key proliferation-related transcription factor, forkhead box protein M1 (FOXM1) is involved in tumorigenesis. Here, we elucidated the underlying mechanism by which HNRNPA2B1-mediated modification of FOXM1 promotes endometrial cancer (EC). The GSE115810 dataset was used to analyze the upregulated gene mRNA in late-stage EC tissues. The expression levels of HNRNPA2B1, FOXM1, and LCN2 in EC samples were shown by western blotting and qPCR. The interaction among HNRNPA2B1, FOXM1, and LCN2 in EC cells was detected using bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down, RNA decay analysis, and luciferase reporter experiments. Cisplatin (DDP)-resistant EC cells were constructed using HEC-1-A and HEC-1-B cells, named HEC-1-A/DDP and HEC-1-B/DDP, respectively. Proliferation, migration, and invasiveness in treated HEC-1-A/DDP and HEC-1-B/DDP cells were detected by EdU, wound healing, and transwell assays. Ferroptosis-resistant gene expression, MDA level, and ROS level were measured. The m6A modification level in EC tissues was elevated. HNRNPA2B1 and FOXM1 levels were upregulated in EC. HNRNPA2B1 expression was positively related to FOXM1 expression in EC samples, and HNRNPA2B1 bound to the 3'UTR of FOXM1 and stabilized FOXM1 mRNA via m6A modification. FOXM1 positively regulated LCN2 expression in EC cells by binding to the LCN2 promotor. Knockdown of FOXM1 downregulated ferroptosis-resistant gene expression and increased MDA and ROS levels in DDP-resistant EC cells. Rescue assays revealed that LCN2 overexpression eliminated the effects mediated by FOXM1 knockdown on the proliferation, migration, invasiveness, and ferroptosis in DDP-resistant EC cells. In conclusion, HNRNPA2B1-mediated mA modification of FOXM1 facilitates drug resistance and inhibits ferroptosis in EC cells by upregulating LCN2 expression.

Gentisic acid mitigates gentamicin-induced nephrotoxicity in rats.

The current investigation was considered to evaluate the beneficial effects of gentisic acid (GA) on gentamicin (GEN)-induced nephrotoxicity in rat kidneys through assessment of oxidative stress, inflammatory cytokines, and histopathological changes. Rats were split into five equal groups. Rats were treated with GA (25, 50, and 100 mg/kg/day, p.o.) for 14 consecutive days and GEN (100 mg/kg, i.p.) was administrated from day 8 to day 14 of the experiment. On the 15th day, blood samples were collected to determine neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), blood urea nitrogen (BUN), and creatinine (Cr) levels. Malondialdehyde (MDA), glutathione (GSH), tumor necrosis factor-alpha (TNF-α) and interleukin-1ß (IL-1ß), and nitric oxide (NO) levels and the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were assessed in the renal tissue. Histopathological evaluations were done to confirm the biochemical results. GEN increased the levels of NGAL, KIM-1, BUN, and Cr in serum as well as MDA, NO, GSH, TNF-α, and IL-1ß in renal tissue. Moreover, GEN administration reduced the activity of CAT, SOD, and GPx in renal tissue. Nonetheless, the administration of GA before and alongside GEN mitigated these deleterious effects. In conclusion, GA has a beneficial effect on biochemical, inflammatory, and oxidative stress indices against GEN-induced nephrotoxicity.

[Notoginsenoside R_1 interferes with renal oxidative stress and Nrf2/HO-1 signaling pathway to alleviate kidney injury in mice with IgA nephropathy and its mechanism].

The purpose of this study was to investigate the effect of notoginsenoside R_1(NGR_1) on alleviating kidney injury by regulating renal oxidative stress and the Nrf2/HO-1 signaling pathway in mice with IgA nephropathy(IgAN) and its mechanism. The mouse model of IgAN was established using a variety of techniques, including continuous bovine serum albumin(BSA) gavage, subcutaneous injections of carbon tetrachloride(CCl_4) castor oil, and tail vein injections of lipopolysaccharide(LPS). After successful modeling, mice with IgAN were randomly separated into a model group, low, medium, and high-dose NGR_1 groups, and a losartan group, and C57BL6 mice were utilized as normal controls. The model and normal groups were given phosphate buffered saline(PBS) by gavage, the NGR_1 groups were given varying dosages of NGR_1 by gavage, and the losartan group was given losartan by gavage for 4 weeks. The 24-hour urine of mice was collected after the last administration, and serum and kidney tissues of mice were taken at the end of the animal experiment. Then urine red blood cell count(URBCC), 24-hour urine protein(24 h protein), serum creatinine(Scr), and blood urea nitrogen(BUN) levels were measured. The enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of galactose-deficient IgA1(Gd-IgA1), kidney injury molecule 1(Kim-1), and neutropil gelatinase-associated lipocalin(NGAL) in the mouse serum. The assay kits were used to detect the levels of malondialdehyde(MDA) and superoxide dismutase(SOD), and immunofluorescence(IF) was used to detect the expression level of glutathione peroxidase 4(GPX4) in the mesangial region. Western blot was used to detect the protein expression of nuclear transcription factor E2 related factor 2(Nrf2)/heme oxygenase 1(HO-1) signaling pathway in the renal tissue. Hematoxylin-eosin(HE) staining was used to observe pathological alterations in the glomerulus of mice. The results revealed that, as compared with the model group, the serum Gd-IgA1 level, URBCC, 24 h protein level, renal damage markers(Kim-1 and NGAL) in the high-dose NGR_1 group decreased obviously and renal function indicators(BUN, Scr) improved significantly. The activity of SOD activity and expression level of GPX4 increased significantly in the high-dose NGR_1 group, whereas the expression level of MDA reduced and protein expression levels of Nrf2 and HO-1 increased. Simultaneously, HE staining of the renal tissue indicated that glomerular damage was greatly decreased in the high-dose NGR_1 group. In conclusion, this study has clarified that NGR_1 may alleviate the kidney injury of mice with IgAN by activating the Nrf2/HO-1 signaling pathway, improving antioxidant capacity, and reducing the level of renal oxidative stress.

Melittin inhibits tumor cell migration and enhances cisplatin sensitivity by suppressing IL-17 signaling pathway gene LCN2 in castration-resistant prostate cancer.

BACKGROUND: Melittin is a small molecule polypeptide extracted from the abdominal cavity of bees, which is used to treat inflammatory diseases and relieve pain. However, the antitumor effect of melittin and its mechanisms remain unclear, especially in castration-resistant prostate cancer (CRPC). METHODS: Through CCK-8 assay, colony formation assay, wound healing assay and Transwell migration assay, we explored the effect of melittin on CRPC cell lines. In addition, with microarray analysis, gene ontology analysis and kyoto encyclopedia of genes and genomes analysis, this study identified key genes and signaling pathways that influence the growth of PC-3 cells. Meanwhile, the effect of melittin on CRPC was also verified through subcutaneous tumor formation experiments. Finally, we also tested the relevant indicators of human prostate cancer (PCa) specimens through immunohistochemistry and H＆E stating. RESULTS: Here, melittin was verified to inhibit the cell proliferation and migration of CPRC. Moreover, RNA-sequence analysis demonstrated that Interleukin-17 (IL-17) signaling pathway gene Lipocalin-2 (LCN2) was downregulated by melittin treatment in CRPC. Further investigation revealed that overexpression of LCN2 was able to rescue tumor suppression and cisplatin sensitivity which melittin mediated. Interestingly, the expression of LCN2 is highly related to metastasis in PCa. CONCLUSIONS: In brief, our study indicates that LCN2 plays an oncogenic role in CRPC and melittin may be selected as an attractive candidate for CRPC therapy.

Vitamin D and calcium co-therapy mitigates pre-established cadmium nephropathy by regulating renal calcium homeostatic molecules and improving anti-oxidative and anti-inflammatory activities in rat.

BACKGROUND: Cadmium (Cd) is a major environmental pollutant and chronic toxicity could induce nephropathy by increasing renal oxidative stress and inflammation. Although vitamin D (VD) and calcium (Ca) prophylactic treatments attenuated Cd-induced cell injury, none of the prior studies measure their renoprotective effects against pre-established Cd-nephropathy. AIMS: To measure the alleviating effects of VD and/or Ca single and dual therapies against pre-established nephrotoxicity induced by chronic Cd toxicity prior to treatment initiation. METHODS: Forty male adult rats were allocated into: negative controls (NC), positive controls (PC), Ca, VD and VC groups. The study lasted for eight weeks and all animals, except the NC, received CdCl2 in drinking water (44 mg/L) throughout the study. Ca (100 mg/kg) and/or VD (350 IU/kg) were given (five times/week) during the last four weeks to the designated groups. Subsequently, the expression of transforming growth factor-ß (TGF-ß1), inducible nitric oxide synthase (iNOS), neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), VD synthesising (Cyp27b1) and catabolizing (Cyp24a1) enzymes with VD receptor (VDR) and binding protein (VDBP) was measured in renal tissues. Similarly, renal expression of Ca voltage-dependent channels (CaV1.1/CaV3.1), store-operated channels (RyR1/ITPR1), and binding proteins (CAM/CAMKIIA/S100A1/S100B) were measured. Serum markers of renal function alongside several markers of oxidative stress (MDA/H2O2/GSH/GPx/CAT) and inflammation (IL-6/TNF-α/IL-10) together with renal cell apoptosis and expression of caspase-3 were also measured. RESULTS: The PC group exhibited hypovitaminosis D, hypocalcaemia, hypercalciuria, proteinuria, reduced creatinine clearance, and increased renal apoptosis/necrosis with higher caspase-3 expression. Markers of renal tissue damage (TGF-ß1/iNOS/NGAL/KIM-1), oxidative stress (MDA/H2O2), and inflammation (TNF-α/IL-1ß/IL-6) increased, whilst the antioxidants (GSH/GPx/CAT) and IL-10 decreased, in the PC group. The PC renal tissues also showed abnormal expression of Cyp27b1, Cyp24a1, VDR, and VDBP, alongside Ca-membranous (CaV1.1/CaV3.1) and store-operated channels (RyR1/ITPR1) and cytosolic Ca-binding proteins (CAM/CAMKIIA/S100A1/S100B). Although VD was superior to Ca monotherapy, their combination revealed the best mitigation effects by attenuating serum and renal tissue Cd concentrations, inflammation and oxidative stress, alongside modulating the expression of VD/Ca-molecules. CONCLUSIONS: This study is the first to show improved alleviations against Cd-nephropathy by co-supplementing VD and Ca, possibly by better regulation of Ca-dependent anti-oxidative and anti-inflammatory actions.

Amyloid ß oligomer promotes microglial galectin-3 and astrocytic lipocalin-2 levels in the hippocampus of mice fed a high-fat diet.

Type 2 diabetes is associated with a risk factor for Alzheimer's disease (AD). Activation of glial cells, such as microglia and astrocytes, is crucial for the development of neuroinflammation in both diabetes and AD. The role of amyloid-beta oligomer (AßO) in the hippocampus of diabetic mice has been investigated; however, the effect of galectin-3 and lipocalin-2 (LCN2) on amyloid toxicity-related glial activation in diabetic mice is not known. To fill this knowledge gap, we fed mice a high-fat diet (HFD) for 20 weeks to induce a diabetic state and then injected the hippocampus with AßO. Sholl analysis of iba-1-positive microglia showed retraction of microglial ramifications in the hippocampus of HFD-fed diabetic mice. AßO treatment caused more retraction of microglial process in HFD-fed mice. In particular, microglial galectin-3 levels and astrocytic LCN2 levels were increased in the hippocampus of HFD-fed mice with AßO treatment. These findings suggest that galectin-3 and LCN2 are involved in amyloid toxicity mechanisms, especially glial activation under diabetic conditions.

Ameliorative effects of glycine on cobalt chloride-induced hepato-renal toxicity in rats.

BACKGROUND: The important roles of liver and kidney in the elimination of injurious chemicals make them highly susceptible to the noxious activities of various toxicants including cobalt chloride (CoCl2 ). This study was designed to investigate the role of glycine in the mitigation of hepato-renal toxicities associated with CoCl2 exposure. METHODS: Forty-two (42) male rats were grouped as Control; (CoCl2 ; 300 ppm); CoCl2 + Glycine (50 mg/kg); CoCl2 + Glycine (100 mg/kg); Glycine (50 mg/kg); and Glycine (100 mg/kg). The markers of hepatic and renal damage, oxidative stress, the antioxidant defense system, histopathology, and immunohistochemical localization of neutrophil gelatinase associated lipocalin (NGAL) and renal podocin were evaluated. RESULTS: Glycine significantly reduced the markers of oxidative stress (malondialdehyde content and H2 O2 generation), liver function tests (ALT, AST, and ALP), markers of renal function (creatinine and BUN), and decreased the expression of neutrophil gelatinase-associated lipocalin (NGAL) and podocin compared with rats exposed to CoCl2 toxicity without glycine treatment. Histopathology lesions including patchy tubular epithelial necrosis, tubular epithelial degeneration and periglomerular inflammation in renal tissues, and severe portal hepatocellular necrosis, inflammation, and duct hyperplasia were observed in hepatic tissues of rats exposed to CoCl2 toxicity, but were mild to absent in glycine-treated rats. CONCLUSION: The results of this study clearly demonstrate protective effects of glycine against CoCl2 -induced tissue injuries and derangement of physiological activities of the hepatic and renal systems in rats. The protective effects are mediated via augmentation of total antioxidant capacity and upregulation of NGAL and podocin expression.

Salvianolic Acid A Improves Rat Kidney Injury by Regulating MAPKs and TGF-ß1/Smads Signaling Pathways.

Salvianolic acid A (SAA) is one of the major components in Salvia miltiorrhiza Bge., with various pharmacological activities, and is likely to be a promising agent for the treatment of kidney diseases. The purpose of this study was to explore the protective effect and mechanisms of SAA on kidney disease. In this study, the improvement effects of SAA (10, 20, 40 mg/kg, i.g.) on kidney injury rats were investigated by detecting the levels of KIM-1, NGAL in serum and UP in the urine of AKI model rats established with gentamicin, as well as the levels of SCr and UREA in serum and IL-6, IL-12, MDA and T-SOD in the kidneys of CKD model rats established with 5/6 nephrectomy. HE and Masson staining were used to observe the histopathological changes in the kidney. Network pharmacology and Western blotting were used to explore the mechanism of SAA in improving kidney injury. The results showed that SAA improved kidney function in kidney injury rats by reducing the kidney index and pathological injury by HE and Masson staining, reducing the levels of KIM-1, NGAL and UP in AKI rats and UREA, SCr and UP in CKD rats, as well as exerting anti-inflammatory and anti-oxidative stress effects by inhibiting the release of IL-6 and IL-12, reducing MDA and increasing T-SOD. Western blotting results showed that SAA significantly reduced the phosphorylation levels of ERK1/2, p38, JNK and smad2/3, and the expression of TLR-4 and smad7. In conclusion, SAA plays a significant role in improving kidney injury in rats and the mechanism may be achieved by regulating the MAPKs and TGF-ß1/smads signaling pathways.

Clemizole hydrochloride, a potent TRPC5 calcium channel inhibitor, prevents cisplatin-induced nephrotoxicity in Spraque-Dawley rats.

Cis-diamminedichloroplatinum (II) (cisplatin, Cis) is widely employed to treat several types of cancer. It has many important toxic side effects; one of the most important of which is nephrotoxicity. Clemizole hydrochloride (Clem) as the most potent inhibitor of TRPC5 channels was tested in an animal model of Cis-induced nephrotoxicity. Rats were divided into the following groups: control; Cis (8 mg/kg); Cis + 1 mg/kg Clem; Cis + 5 mg/kg Clem; Cis + 10 mg/kg Clem. Kidney injury was detected by histopathological and biochemical analysis. Urine urea nitrogen (UUN), creatinine, urine neutrophil gelatinase-associated lipocalin (NGAL), serum catalase (CAT), and malondialdehyde (MDA) levels were determined by enzyme-linked immunosorbent assay. Total antioxidant status (TAS) and total oxidant status (TOS) were studied using a colorimetric assay. Nephrin, synaptopodin, and Rac family small GTPase 1 (RAC1) expressions were detected by Western blot analysis. Cis was found to induce histopathological alterations, including tubular degeneration, congestion, hemorrhage, hyaline casts, glomerular collapse, and apoptotic cell death. Clem at a dose of 1 and 5 mg/kg attenuated histopathological alterations. UUN, creatinine, and NGAL levels increased in the Cis-administered group, while all doses of Clem decreased in those. CAT and TAS levels decreased, while TOS and oxidative stress index levels increased in the Cis-treated group. A dose of 1 and 5 mg Clem showed antioxidant effects against oxidative stress. Cis induced lipid peroxidation by increasing MDA levels. All doses of Clem reduced MDA levels. Nephrin and synaptopodin expressions were decreased by Cis, and all doses of Clem increased that. All doses of Clem successfully depressed RAC1 expression. Clem showed a highly ameliorating effect on toxicity caused by Cis by blocking TRPC5 calcium channels.

Silencing LCN2 suppresses oral squamous cell carcinoma progression by reducing EGFR signal activation and recycling.

BACKGROUND: EGFR is an important signal involved in tumor growth that can induce tumor metastasis and drug resistance. Exploring targets for effective EGFR regulation is an important topic in current research and drug development. Inhibiting EGFR can effectively inhibit the progression and lymph node metastasis of oral squamous cell carcinoma (OSCC) because OSCC is a type of cancer with high EGFR expression. However, the problem of EGFR drug resistance is particularly prominent, and identifying a new target for EGFR regulation could reveal an effective strategy. METHODS: We sequenced wild type or EGFR-resistant OSCC cells and samples from OSCC patients with or without lymph node metastasis to find new targets for EGFR regulation to effectively replace the strategy of directly inhibiting EGFR and exert an antitumor effect. We then investigated the effect of LCN2 on OSCC biological abilities in vitro and in vivo through protein expression regulation. Subsequently, we elucidated the regulatory mechanism of LCN2 through mass spectrometry, protein interaction, immunoblotting, and immunofluorescence analyses. As a proof of concept, a reduction-responsive nanoparticle (NP) platform was engineered for effective LCN2 siRNA (siLCN2) delivery, and a tongue orthotopic xenograft model as well as an EGFR-positive patient-derived xenograft (PDX) model were applied to investigate the curative effect of siLCN2. RESULTS: We identified lipocalin-2 (LCN2), which is upregulated in OSCC metastasis and EGFR resistance. Inhibition of LCN2 expression can effectively inhibit the proliferation and metastasis of OSCC in vitro and in vivo by inhibiting EGFR phosphorylation and downstream signal activation. Mechanistically, LCN2 binds EGFR and enhances the recycling of EGFR, thereby activating the EGFR-MEK-ERK cascade. Inhibition of LCN2 effectively inhibited the activation of EGFR. We translated this finding by systemic delivery of siLCN2 by NPs, which effectively downregulated LCN2 in the tumor tissues, thereby leading to a significant inhibition of the growth and metastasis of xenografts. CONCLUSIONS: This research indicated that targeting LCN2 could be a promising strategy for the treatment of OSCC.

Reduced secretion of LCN2 (lipocalin 2) from reactive astrocytes through autophagic and proteasomal regulation alleviates inflammatory stress and neuronal damage.

LCN2/neutrophil gelatinase-associated lipocalin/24p3 (lipocalin 2) is a secretory protein that acts as a mammalian bacteriostatic molecule. Under neuroinflammatory stress conditions, LCN2 is produced and secreted by activated microglia and reactive astrocytes, resulting in neuronal apoptosis. However, it remains largely unknown whether inflammatory stress and neuronal loss can be minimized by modulating LCN2 production and secretion. Here, we first demonstrated that LCN2 was secreted from reactive astrocytes, which were stimulated by treatment with lipopolysaccharide (LPS) as an inflammatory stressor. Notably, we found two effective conditions that led to the reduction of induced LCN2 levels in reactive astrocytes: proteasome inhibition and macroautophagic/autophagic flux activation. Mechanistically, proteasome inhibition suppresses NFKB/NF-κB activation through NFKBIA/IκBα stabilization in primary astrocytes, even under inflammatory stress conditions, resulting in the downregulation of Lcn2 expression. In contrast, autophagic flux activation via MTOR inhibition reduced the intracellular levels of LCN2 through its pre-secretory degradation. In addition, we demonstrated that the N-terminal signal peptide of LCN2 is critical for its secretion and degradation, suggesting that these two pathways may be mechanistically coupled. Finally, we observed that LPS-induced and secreted LCN2 levels were reduced in the astrocyte-cultured medium under the above-mentioned conditions, resulting in increased neuronal viability, even under inflammatory stress.Abbreviations: ACM, astrocyte-conditioned medium; ALP, autophagy-lysosome pathway; BAF, bafilomycin A1; BTZ, bortezomib; CHX, cycloheximide; CNS, central nervous system; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; JAK, Janus kinase; KD, knockdown; LCN2, lipocalin 2; LPS, lipopolysaccharide; MACS, magnetic-activated cell sorting; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MTOR, mechanistic target of rapamycin kinase; NFKB/NF-κB, nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105; NFKBIA/IκBα, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha; OVEX, overexpression; SLC22A17, solute carrier family 22 member 17; SP, signal peptide; SQSTM1, sequestosome 1; STAT3, signal transducer and activator of transcription 3; TNF/TNF-α, tumor necrosis factor; TUBA, tubulin, alpha; TUBB3/ß3-TUB, tubulin, beta 3 class III; UB, ubiquitin; UPS, ubiquitin-proteasome system.

Lipocalin 2, synthesized using a cell-free protein synthesis system and encapsulated into liposomes, inhibits the adhesion of Porphyromonas gingivalis to human oral epithelial cells.

BACKGROUND AND OBJECTIVE: Lipocalin 2 (LCN2), a glycoprotein expressed in epithelial cells and leukocytes, has an antibacterial effect and plays a role in innate immunity. The delivery of LCN2 encapsulated in liposomes to oral epithelium may be useful to prevent oral infectious diseases. This study aimed to investigate the inhibitory effect of LCN2, artificially synthesized using a cell-free protein synthesis (CFPS) system, on the adhesion of Porphyromonas gingivalis to oral epithelial cells in order to approach oral healthcare using LCN2. METHODS: LCN 2 was synthesized using a CFPS system and assayed by Western blotting, mass spectrometry and enzyme-linked immunosorbent assay (ELISA). The bilayer liposomes were prepared by the spontaneous transfer method using 1,2-dioleoyl-sn-glycero-3 phosphocholine (DOPC), 3-sn-phosphatidylcholine from Egg Yolk (Egg-PC), and 1,2-dioleoyl-sn-glycero-3 phosphoethanolamine (DOPE). The cellular and medium fractions derived from the culture of oral epithelial cells with liposome-encapsulated LCN2 were assayed by Western blotting and ELISA. The effect of the synthesized LCN2 on adhesion of the labeled P. gingivalis to oral epithelial cells was investigated as an evaluation of its antibacterial activity. RESULTS: The synthesized LCN2 protein was identified by Western blotting; its amino acid sequence was similar to that of recombinant LCN2 protein. The additions of DOPE and octa-arginine in the outer lipid-layer components of liposome significantly increased the delivery of liposomes to epithelial cells. When oral epithelial cells were cultured with the synthesized and liposome-encapsulated LCN2, LCN2 was identified in the cellular and medium fractions by Western blotting and its concentration in the cellular fraction from the culture with the synthesized LCN2 was significantly higher than that of a template DNA-free protein. The synthesized LCN2 and liposome-encapsulated LCN2 significantly inhibited the adhesion of P. gingivalis to oral epithelial cells compared with template DNA-free protein. CONCLUSION: LCN2 was artificially synthesized by a CFPS system, encapsulated in liposomes, and delivered to oral epithelial cells, and demonstrated an antibacterial action against P. gingivalis. This approach may become a useful model for oral healthcare.

Does G Protein-Coupled Estrogen Receptor 1 Contribute to Cisplatin-Induced Acute Kidney Injury in Male Mice?

Nephrotoxicity is the dose-limiting side-effect of the chemotherapeutic agent cisplatin (Cp). Recent evidence points to renal protective actions of G protein-coupled estrogen receptor 1 (GPER1). In addition, it has been shown that GPER1 signaling elicits protective actions against acute ischemic injuries that involve multiple organ systems; however, the involvement of GPER1 signaling in Cp-induced acute kidney injury (AKI) remains unclear. This study tested whether genetic deletion of GPER1 exacerbates Cp-induced AKI in male mice. We subjected male mice, homozygous (homo) and heterozygous (het) knockout for the GPER1 gene, and wild-type (WT) littermates to Cp or saline injections and assessed markers for renal injury on the third day after injections. We also determined serum levels of proinflammatory markers in saline and Cp-treated mice. Given the protective role of heme oxygenase-1 (HO-1) in Cp-mediated apoptosis, we also investigated genotypic differences in renal HO-1 abundance, cell death, and proliferation by Western blotting, the TUNEL assay, and Ki67 immunostaining, respectively. Cp increased serum creatinine, urea, and neutrophil gelatinase-associated lipocalin (NGAL) levels, the renal abundance of kidney injury molecule-1, and NGAL in all groups. Cp-induced AKI resulted in comparable histological evidence of injury in all genotypes. WT and homo mice showed greater renal HO-1 abundance in response to Cp. Renal HO-1 abundance was lower in Cp-treated homo, compared to Cp-treated WT mice. Of note, GPER1 deletion elicited a remarkable increase in renal apoptosis; however, no genotypic differences in cell proliferation were observed. Cp augmented kidney Ki67-positive counts, regardless of the genotype. Overall, our data do not support a role for GPER1 in mediating Cp-induced renal injury. GPER1 deletion promotes renal apoptosis and diminishes HO-1 induction in response to Cp, suggesting that GPER1 may play cytoprotective and anti-apoptotic actions in AKI. GPER1-induced regulation of HO-1 and apoptosis may offer novel therapeutic targets for the treatment of AKI.

The effect of glucagon like peptide-1 receptor agonist on behavioral despair and anxiety-like behavior in ovariectomized rats: Modulation of BDNF/CREB, Nrf2 and lipocalin 2.

Ovariectomized (OVX) rodents show behavioral despair and anxiety-like behaviors. Glucagon-like peptide-1 receptor agonists (GLP-1RA) possess neuroprotective effects by reducing oxidative stress and neuroinflammation, thereby preventing synaptic loss. The objective of the present study is to evaluate the effect of GLP-1RA, namely liraglutide, on emotional behaviors, and to identify the level of oxidative stress, neuroinflammation, and BDNF signaling in the hippocampus of OVX rats. Forty female young Wistar rats were divided into 5 groups: Control, Control+liraglutide treated, OVX, OVX+fluoxetine, and OVX+liraglutide (150 µg/kg for 15 days, sc). Open field test and elevated plus-maze test were used to evaluate behaviors that are suggestive of anxiety. A forced swimming test was used to evaluate behavioral despair. At the end of the experiments, blood glucose level and body weight gain were measured. The levels of BDNF, CREB, Nrf2, and lipocalin 2 in the hippocampal tissue were measured by ELISA. Malondialdehyde (MDA) and glutathione levels were also evaluated. Statistical analysis was conducted through ANOVA and Bonferroni tests. Seven weeks post-OVX rats exhibited high anxiety related behavior and behavioral despair in comparison with the control groups. These behavioral changes were associated with increased lipocalin 2 and MDA levels in rats. Moreover, BDNF, CREB, and Nrf2 levels decreased significantly in the hippocampus of OVX rats. Liraglutide treatment limited the reduction of BDNF and Nrf2 levels in the hippocampus, maintaining them at the control levels. Liraglutide treatment also prevented the symptoms of behavioral despair and anxiety related behavior. As the main finding of the study GLP-1RA reduced behavioral despair and anxiety level and this may be related to the preservation of BDNF/Nrf2 levels and the decrease in oxidative stress and lipocalin 2 levels in the hippocampus.

N-acetylcysteine Ameliorates Vancomycin-induced Nephrotoxicity by Inhibiting Oxidative Stress and Apoptosis in the in vivo and in vitro Models.

Background: Oxidative stress-related apoptosis is considered as the key mechanism implicated in the pathophysiology of nephrotoxicity with vancomycin (VCM) therapy. We evaluated the possible effects of N-acetylcysteine (NAC) on VCM-induced nephrotoxicity and the underlying mechanism. Methods: VCM-induced nephrotoxicity was established using HK-2 cells and SD rats and observed by measuring cell survival, kidney histological changes, renal function and kidney injury related markers (KIM-1 and NGAL). Oxidative stress, renal cell apoptosis and the involved signaling pathways were also evaluated. Results: In model rats, NAC could protect against VCM-induced acute kidney injury with histological damage, renal dysfunction, and increased Cre and BUN levels. In HK-2 cells, VCM-induced decreased cell viability was restored by NAC. In addition, increased expression of caspase-3, KIM-1 and NGAL suffering from VCM was also reversed by NAC in vivo and in vitro. NAC inhibited ROS production, decreased cell apoptosis by decreasing the Bax/Bcl-2 ratio and caspase-3 expression in HK-2 cells and regulated oxidative stress indicators in the kidney by decreasing GSH, SOD and CAT activity and increasing MDA levels. Furthermore, NAC could effectively reverse VCM-associated increased P38 MAPK/JNK phosphorylation. Conclusions: The results demonstrated that NAC had a protective effect against nephrotoxicity from VCM by inhibiting oxidative stress and apoptosis via P38 MAPK/JNK.

Apigenin attenuates molecular, biochemical, and histopathological changes associated with renal impairments induced by gentamicin exposure in rats.

Gentamicin (GM) is an aminoglycoside antibiotic used to treat bacterial infections. However, its application is accompanied by renal impairments. Apigenin is a flavonoid found in many edible plants with potent therapeutic values. This study was designed to elucidate the therapeutic effects of apigenin on GM-induced nephrotoxicity. Animals were injected orally with three different doses of apigenin (5 mg kg-1 day-1, 10 mg kg-1 day-1, and 20 mg kg-1 day-1). Apigenin administration abolished the alterations in the kidney index and serum levels of kidney-specific functions markers, namely blood urea nitrogen and creatinine, and KIM-1, NGAL, and cystatin C following GM exposure. Additionally, apigenin increased levels of enzymatic (glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase) and non-enzymatic antioxidant proteins (reduced glutathione) and decreased levels of lipid peroxide, nitric oxide, and downregulated nitric oxide synthase-2 in the kidney tissue following GM administration. At the molecular scope, apigenin administration was found to upregulate the mRNA expression of Nfe2l2 and Hmox1 in the kidney tissue. Moreover, apigenin administration suppressed renal inflammation and apoptosis by decreasing levels of interleukin-1ß, tumor necrosis factor-alpha, nuclear factor kappa-B, Bax, and caspase-3, while increasing B-cell lymphoma-2 compared with those in GM-administered group. The recorded data suggests that apigenin treatment could be used to alleviate renal impairments associated with GM administration.

Protective effects of p-coumaric acid against gentamicin-induced nephrotoxicity in rats.

The most important side effect of gentamicin (GM) is nephrotoxicity. p-Coumaric acid (PCA) is a phenolic compound that scavenges free radicals, reduces fibrosis, and tissue damage. This study investigates the protective effect of PCA on tissue damage and kidney function in gentamicin-induced nephrotoxicity (GIN). Thirty-five rats were separated into five groups and each group contained seven animals: control group, ethanol group, GM group, PCA group, and GM + PCA group. At the end of the seven-day treatment, the rats were sacrificed after blood and kidney tissue samples were taken. While serum urea, creatinine, and neutrophil gelatinase-associated lipocalin (NGAL) levels increased significantly in the GM group compared to the control, they showed a significant decrease in the GM + PCA group compared to the GM. Serum tumor necrosis factor-α (TNF-α) and tissue malondialdehyde (MDA) levels were significantly increased in the GM group compared to the control. While the tissue total oxidant status (TOS) and oxidative stress index (OSI) values of the GM group were significantly higher than the control, they showed a significant decrease in the GM + PCA group compared to the GM. In the histopathological examination, significant tubular necrosis and tubulointerstitial inflammation were detected in the proximal tubules in the GM group compared to the control, while a significant decrease was observed in the severity of these findings in the GM + PCA group compared to the GM. This study shows that PCA has biochemical and histopathological ameliorating effects on GIN in the rat model.

Electroacupuncture improves repeated social defeat stress-elicited social avoidance and anxiety-like behaviors by reducing Lipocalin-2 in the hippocampus.

BACKGROUND: Post-traumatic stress disorder (PTSD) is a trauma-related disorder that is associated with pro-inflammatory activation and neurobiological impairments in the brain and leads to a series of affective-like behaviors. Electroacupuncture (EA) has been proposed as a clinically useful therapy for several brain diseases. However, the potential role of EA treatment in PTSD and its molecular and cellular mechanisms has rarely been investigated. METHODS: We used an established preclinical social defeat stress mouse model to study whether EA treatment modulates PTSD-like symptoms and understand its underlying mechanisms. To this end, male C57BL/6 mice were subjected to repeated social defeat stress (RSDS) for 6 consecutive days to induce symptoms of PTSD and treated with EA at Baihui (GV 20) and Dazhui (GV 14) acupoints. RESULTS: The stimulation of EA, but not needle insertion at Baihui (GV 20) and Dazhui (GV 14) acupoints effectively improved PTSD-like behaviors such as, social avoidance and anxiety-like behaviors. However, EA stimulation at the bilateral Tianzong (SI11) acupoints did not affect the PTSD-like behaviors obtained by RSDS. EA stimulation also markedly inhibited astrocyte activation in both the dorsal and ventral hippocampi of RSDS-treated mice. Using next-generation sequencing analysis, our results showed that EA stimulation attenuated RSDS-enhanced lipocalin 2 expression in the hippocampus. Importantly, using double-staining immunofluorescence, we observed that the increased lipocalin 2 expression in astrocytes by RSDS was also reduced by EA stimulation. In addition, intracerebroventricular injection of mouse recombinant lipocalin 2 protein in the lateral ventricles provoked social avoidance, anxiety-like behaviors, and the activation of astrocytes in the hippocampus. Interestingly, the overexpression of lipocalin 2 in the brain also altered the expression of stress-related genes, including monoamine oxidase A, monoamine oxidase B, mineralocorticoid receptor, and glucocorticoid receptor in the hippocampus. CONCLUSIONS: This study suggests that the treatment of EA at Baihui (GV 20) and Dazhui (GV 14) acupoints improves RSDS-induced social avoidance, anxiety-like behaviors, astrocyte activation, and lipocalin 2 expression. Furthermore, our findings also indicate that lipocalin 2 expression in the brain may be an important biomarker for the development of PTSD-related symptoms.