Daily Intraperitoneal Injections Research Articles

Ras guanine nucleotide-releasing protein-4 promotes renal inflammatory injury in type 2 diabetes mellitus

IntroductionRas guanine nucleotide-releasing protein-4 (RasGRP4) is an activator of Ras protein, which plays significant roles in both the inflammatory response and immune activation. This study determined the role of RasGRP4 in diabetic kidney disease (DKD) progression. MethodsCRISPR/Cas9 technology was used to establish RasGRP4 knockout (KO) mice. Diabetes was induced by a high-fat diet combined with five consecutive daily intraperitoneal injections of streptozotocin (60 mg/kg) in C57BL/6J wild-type (WT) mice and RasGRP4 KO mice. Hematoxylin and eosin, periodic acid-Schiff, and Masson's trichrome staining were used to observe the histology of pathological injury. Immunohistochemical staining was used to analyze inflammatory cell infiltration. Quantitative PCR and Western blotting were used to detect the expression of inflammatory mediators and the activation of signaling pathways in renal tissues. In vitro cell co-culture experiments were performed to explore the interactions between peripheral blood mononuclear cells (PBMCs) and glomerular endothelial cells (GEnCs). ResultsRasGRP4 KO mice developed less severe diabetic kidney injury compared to WT mice, exhibiting lower proteinuria, reduced CD3+ T lymphocyte and F4/80+ macrophage infiltration, less inflammatory mediator expression including interleukin 6, tumor necrosis alpha, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, and lower expression levels of critical signal transduction molecules in the NLR family pyrin domain-containing 3 inflammasome and mitogen-activated protein kinase (MAPK)/nuclear factor kappa B (NF-κB) signaling pathways in the diabetic kidney. In vitro experiments showed that the adhesion function of PBMCs of RasGRP4 KO mice was reduced compared to that of WT mice. Moreover, the expression of adhesion molecules and critical signal transduction molecules in the NLRP3 inflammasome and MAPK/NF-κB signaling pathways in GEnCs was stimulated by the supernatant of PBMCs, which were derived from RasGRP4 KO mice treated with high glucose and were also significantly reduced compared to those derived from WT mice. ConclusionRasGRP4 promotes the inflammatory injury mediated by PBMCs in diabetes, probably by regulating the interaction between PBMCs and GEnCs and further activating the NLRP3 inflammasome and MAPK/NF-κB signaling pathways.

Exogenous H2S Ameliorates High Salt-Induced Hypertension by Alleviating Oxidative Stress and Inflammation in the Paraventricular Nucleus in Dahl S Rats

Hydrogen sulfide (H2S) is an important gaseous signaling molecule that regulates cardiovascular activity in animals. The hypothalamic paraventricular nucleus (PVN) is a major integrative region involved in blood pressure (BP) regulation. We explored whether exogenous H2S application by intraperitoneal injection of sodium hydrosulfide (NaHS) alleviates BP increase induced by a high salt diet (HSD) and the role of PVN in Dahl salt-sensitive (Dahl S) rats. Dahl S rats were divided into four groups according to diet regime (normal salt diet [NSD] and HSD) and treatment method (daily intraperitoneal NaHS or saline injection). We monitored BP, food and water intake, and body weight for 8 weeks. Plasma, kidney, and brain tissues were collected at the end of the experiment. We found that exogenous H2S not only delayed BP elevation but also attenuated the increase in the levels of norepinephrine, cystatin C, and blood urea nitrogen in the plasma of Dahl S rats with an HSD. Furthermore, H2S enhanced the total antioxidant capacity, superoxide dismutase, and glutathione peroxidase in the PVN. Exogenous H2S attenuated the protein expression of the nuclear factor-κB pathway and proinflammatory cytokines, which were significantly higher in the PVN in rats with an HSD than in rats with an NSD. Additionally, exogenous H2S relieved PVN neuronal apoptosis induced by an HSD. These findings suggest that exogenous H2S attenuates hypertension caused by an HSD by ameliorating oxidative stress, inflammation, and apoptosis in the PVN. This study provides evidence of the benefits of peripheral H2S therapy for hypertension.

Leonurine attenuates cisplatin nephrotoxicity by suppressing the NLRP3 inflammasome, mitochondrial dysfunction, and endoplasmic reticulum stress.

Cisplatin has been widely accepted as an effective chemotherapy drug with various side effects, including nephrotoxicity. The mechanisms of cisplatin-induced acute kidney injury (AKI) are complex, and there are limited renoprotective approaches. Leonurine is the main active compound of a Chinese herb and has recently been reported to have a protective effect on the kidneys. This study aimed to verify the renoprotective effect of leonurine in attenuating cisplatin-induced AKI and explore the potential associated mechanisms. C57BL/6 mice were divided into four groups (Sham, Cisplatin, Leonurine, and Cisplatin + Leonurine). Mice in the leonurine-treated groups were pretreated with a daily intraperitoneal injection of 25mg/kg leonurine. AKI was induced by injecting cisplatin once intraperitoneally at 20mg/kg body weight. Mice were killed on day 5. Kidney injury was assessed using a serum biochemical and histological assay. Apoptosis was evaluated using a terminal deoxyribonucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining assay and Western blot. Antioxidant enzymes were detected using commercial kits. The improvement in inflammasome activation, mitochondrial dysfunction, and endoplasmic reticulum stress (ERS) were assessed by polymerase chain reaction (PCR) and Western blot, respectively. Leonurine treatment improved kidney function by preventing renal tubular injury and apoptosis. Expression of nucleotide-binding leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome components and inflammatory cytokines, mitochondrial dysfunction, and ERS were all alleviated by leonurine. The results indicate that leonurine plays a protective role in cisplatin-induced AKI and may represent an effective multi-targeted intervention strategy.

Restoring erectile function by combined treatment with JNK inhibitor and HDAC inhibitor in a rat model of cavernous nerve injury.

The main pathophysiologic conditions of erectile dysfunction (ED) after radical prostatectomy are considered to be corporal fibrosis and apoptosis induced by cavernosal nerve (CN) injury. In a rat model of CN crush injury (CNCI), we investigated whether combination treatment with JNK inhibitor (JNKi), SP600125, and HDAC inhibitor (HDACi), suberoylanilide-hydroxamic-7 acid (SAHA), for 2weeks after CNCI would restore erectile function by suppressing fibrosis and apoptosis through normalization of JNK and HDAC pathways. Seventy 12-week-old rats were randomly divided into five groups: Sham surgery, CNCI alone, CNCI treated with daily intraperitoneal injection of 10mg/kg JNKi, CNCI treated with daily oral administration of 25.0mg/kg HDACi, and CNCI daily treated with a combination. Two weeks after CNCI, we investigated the erectile response to electrostimulation and conducted histological staining, caspase-3 activity assay, and western blot analysis. CNCI alone resulted in significantly reduced intracavernosal pressure/mean arterial pressure (MAP) and area under the curve/MAP, decreased smooth muscle (SM)/collagen ratio and SM content, higher caspase-3 activity, and increased protein levels of total HDAC3, transforming growth factor (TGF)-β, fibronectin, and c-Jun phosphorylation, compared with the Sham surgery. The CNCI groups exposed to JNKi, HDACi or both showed improvements in erectile-responses and SM/collagen ratio, compared to the CNCI alone. The combined treatment showed additional improvement in erectile responses at 1.0V stimulation and in SM/collagen ratio compared to the single agent treatment. SM content, caspase-3 activity, and c-Jun phosphorylation improved in the two CNCI groups exposed to JNKi. The two CNCI groups exposed to HDACi showed normalization of protein levels of HDAC3, fibronectin, and TGF-β. The combined administration of JNKi and HDACi during the acute phase after CNCI in rats can preserve ED by suppressing cavernosal fibrosis and apoptosis by normalizing the HDAC/TGF-β and JNK pathways.

Tumor treating fields (TTFields) in combination with sorafenib inhibit hepatocellular carcinoma in vitro and in vivo.

464 Background: Hepatocellular carcinoma (HCC) is a highly malignant liver cancer and a leading cause of cancer related mortality. Sorafenib was the first approved systemic treatment for HCC, and remains one of few front-line treatments for this malignancy. Tumor treating fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields that exert antimitotic effects on cancerous cells. Results of the phase 2 HEPANOVA study of TTFields (150 kHz) plus sorafenib for advanced HCC support investigation of TTFields in a randomized controlled phase 3 study. The current research aimed to describe the in vitro and in vivo efficacy of this combination and to elucidate details regarding the underlying mechanism of action. Methods: In vitro examinations were performed in HepG2 and Huh-7D12 human HCC cell lines, to which TTFields at a frequency of 150 kHz were applied using the inovitro system. Autophagy was examined by western blot and fluorescence detection of microtubule-associated protein light chain 3 (LC3) levels, an accepted autophagy marker. The effect of TTFields in combination with sorafenib was evaluated using cytotoxic, clonogenic, and apoptotic assays. In vivo, SD rats were inoculated orthotopically into the left hepatic lobe with N1S1 HCC cells. 7 days later, TTFields or sham (heat) were applied to the abdominal region of the rats, continuously for 6 days. Daily intraperitoneal injections of sorafenib (10 mg/kg/day) or vehicle were performed during this time. To determine tumor volume growth, MRI images were acquired before and after treatment. Levels of autophagy and apoptosis were examined in tumor sections by immunohistochemistry for LC3 and cleaved PARP, respectively. Results: Application of TTFields induced autophagy in HCC cells. TTFields delivery was cytotoxic to the cells, reduced their colony forming ability, and induced apoptosis while combination with sorafenib elevated these effects. In vivo, tumor volume increased 6-fold in control animals vs 1.6-fold in animals treated with TTFields plus sorafenib. This effect was accompanied by significantly elevated levels of cleaved PARP and LC3 within the tumors of treated relative to control rats. Conclusions: The results demonstrate induction of autophagy and apoptosis in HCC following treatment with TTFields. Concomitant application of TTFields with sorafenib enhanced efficacy via a mechanism that may involve overwhelming autophagy, in vitro and in vivo.

Therapeutic Potential of Combined Therapy of Vitamin A and Vitamin C in the Experimental Autoimmune Encephalomyelitis (EAE) in Lewis Rats.

Demyelination, inflammation, oxidative injury, and glial activation are the main pathological hallmarks of multiple sclerosis (MS). Vitamins, as essential micronutrients, seem to be crucial in the pathogenesis of MS, and particularly vitamins A and C were found to have a protective role in MS development or progression. In this study, the therapeutic potential of combined therapy of vitamins A and C on progression of experimental autoimmune encephalomyelitis (EAE) and myelin repair mechanisms was examined. EAE, an animal model of MS, was induced in female Lewis rats. The rats were treated with daily intraperitoneal injections of vitamins A and C and their combination. We found that co-supplementation of vitamins A and C mitigated neurological severity and EAE disease progression. Histological study confirmed a significant reduction in demyelination size, inflammation and immune cell infiltration as well as microglia and astrocyte activation following co-administration of vitamins A and C. Co-administration of vitamins A and C also decreased the levels of pro-inflammatory cytokines (TNF-α, IL1β) and iNOS and increased gene expressions of IL-10, Nrf-2, HO-1, and MBP. Combination therapy of vitamins A and C also increased the total antioxidant capacity and decreased levels of oxidative stress markers. Finally, we proved that co-administration of vitamins A and C has anti-apoptotic and neuroprotective impacts in EAE via decreasing caspase-3 and increasing BDNF and NeuN expressing cells. The present study suggests that combined therapy of vitamins A and C may be an effective strategy for development of alternative medicine in boosting myelin repair in demyelinating diseases.

The effect of progesterone administration on the expression of metastasis tumor antigens (MTA1 and MTA3) in placentas of normal and dexamethasone-treated rats.

Dexamethasone (DEX) induces intrauterine growth restriction (IUGR)in pregnant rats. IUGR can occur due to apoptosis of trophoblasts, which is believed to be inhibited by progesterone (P4). A group of genes called MTAs play a role in proliferation and apoptosis. MTA1 upregulates trophoblasts proliferation and differentiation, while MTA3 downregulates proliferation and induces apoptosis. Hence, we hypothesized that during IUGR, placental MTA1 decreases and MTA3 increases and this is reversed by P4 treatment. Pregnant Sprague-Dawley rats were divided into 4 groups based on daily intraperitoneal injections: control (C, saline), DEX (DEX, 0.2mg/kg/day), DEX and P4 (DEX + P4, DEX: 0.2mg/kg/day, P4: 5mg/kg/day) and P4-treated (P4, 5mg/kg/day) groups. Injections were started on 15 dg until the day of dissection (19 or 21 dg). Gene and protein expressions of MTA1 and MTA3 were studied in the labyrinth (LZ) and basal (BZ) zones using real-time PCR and Western blotting, respectively. DEX treatment induced 18% reduction in fetal body weight (p < 0.001) and 30% reduction in placental weight (p < 0.01). Maternal P4 level was also significantly lower in DEX treated groups (p < 0.05). MTA1 expression was decreased in the LZ (gene, p < 0.001) and BZ (protein p < 0.01), while MTA3 protein expression was upregulated in the LZ with DEX treatment (p < 0.001). These changes were reversed with P4 treatment. The findings of the present study indicate that DEX induces IUGR through changing the expression of placental MTA1 and MTA3 antigens and P4 improved pregnancy outcome by preventing the changes in MTAs expression.

Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation

BackgroundThe brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial–glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types.MethodsMouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function.ResultsHerein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood–brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function.ConclusionsAgo2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

Therapeutic Effect of Berberine Versus Methotrexate on Histopathology in a Rat Model of Pristane-Induced Arthritis

Introduction: Treatment options for rheumatoid arthritis have potentially fatal adverse effects and failure to achieve complete cure. Alternative medicines are, therefore, being researched for this purpose. Berberine is one of such compounds with high antioxidant activity that may prove beneficial in this disease.&#x0D; Aims &amp; Objectives: To compare effects of berberine with methotrexate on pristane induced arthritis in rats.&#x0D; Place and duration of study: Post Graduate Medical Institute, Lahore; March to May 2014.&#x0D; Material &amp; Methods: Forty female Sprague Dawley rats were allotted to five groups including a berberine control. Arthritis developed in 14 days with a single intradermal injection of pristane in arthritis control and experimental groups. Starting Day 15, berberine and methotrexate were administered as single daily intraperitoneal injection for next 14 days. Arthritis resolution was assessed by measuring body weight, clinical score of arthritis on day 0, 14 and 28 and joint histopathology terminally. Data was analyzed using SPSS version 20, p value &lt;0.05 was considered significant.&#x0D; Results: Arthritis induction reduced body weight in pristane administered groups (142.87 ± 3.56, 146.25 ± 7.49, 112.37 ± 6.23, 114.50 ±3.85, 113.62 ± 7.72 g in Group I, II, III, IV and V respectively) at day 14. Berberine and methotrexate treatment restored body weight in comparison to continuous loss in arthritis control animals on day 28 (155.87 ± 3.72,&#x0D; 162.00 ± 7.96, 105.25 ± 8.04, 133.75 ± 4.89, 133.12 ± 9.24 g in Group I, II, III, IV and V respectively). Berberine and methotrexate both reduced joint inflammation (clinical score 15±1.51, 6.75±1.48, 3.25±1.48 in arthritis control, berberine and methotrexate treated groups). Methotrexate was, however, more effective in reducing clinical arthritis score than berberine on day 28 (p value &lt;0.001). Histopathological changes were reversed similarly by both drugs.&#x0D; Conclusion: Berberine is effective in treating rheumatoid arthritis though less than methotrexate.

L-Carnitine improves follicular survival and function in ovarian grafts in the mouse.

Ovarian tissue transplantation is performed to preserve fertility in patients undergoing chemotherapy and radiotherapy. However, the ischemia-reperfusion injury which occurs after the ovarian tissue transplantation causes follicular depletion and apoptosis. l -Carnitine has antioxidant and anti-inflammation properties. Therefore, we aimed to investigate the beneficial effect of l -carnitine on mouse ovaries following heterotopic autotransplantation. Mice were randomly divided into three groups (six mice per group): control, autografted and autografted+l -carnitine (200mg/kg daily intraperitoneal injections). Seven days after ovary autografting, the serum levels of malondialdehyde (MDA), total antioxidant capacity, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-10 were measured. Ovary histology, serum concentrations of progesterone and estradiol were also measured 28days after autotransplantation. Data were analysed using one-way analysis of variance (ANOVA) and Tukey test, and the means were considered significantly different at P Key results: In the autografted+l -carnitine group, the total volume of the ovary, the volume of the cortex, the number of follicles, the serum concentrations of IL-10, estradiol and progesterone significantly increased compared to the autografted group. In the autografted+l -carnitine group, serum concentrations of IL-6, TNF-α and MDA were significantly decreased compared to the autografted group. Our results indicated that l -carnitine can ameliorate the consequences of ischemia-reperfusion on the mice ovarian tissue following autotransplantation. l -carnitine improves the structure and function of transplanted ovaries.

Drag-Reducing Polymers Improve Vascular Hemodynamics and Tissue Oxygen Supply in Mouse Model of Diabetes Mellitus.

Diabetes mellitus (DM) is a chronic metabolic disease characterised by hyperglycaemia and glucose intolerance caused by impaired insulin action and/or defective insulin secretion. Long-term hyperglycaemia leads to various structural and functional microvascular changes within multiple tissues, including the brain, which involves blood-brain barrier alteration, inflammation and neuronal dysfunction. We have shown previously that drag-reducing polymers (DRP) improve microcirculation and tissue oxygen supply, thereby reducing neurologic impairment in different rat models of brain injury. We hypothesised that DRP could improve cerebral and skin microcirculation in the situation of progressive microangiopathies associated with diabetes using a mouse model of diabetes mellitus. Diabetes was induced in C57BL/6 J mice with five daily consecutive intraperitoneal injections of streptozotocin (50mg/kg/day). Animals with plasma glucose concentrations greater than 250mg/dL were considered diabetic and were used in the study following four months of diabetes. DRP (2ppm) was injected biweekly during the last two weeks of the experiment. Cortical and skin (ear) microvascular cerebral blood flow (mCBF) and tissue oxygen supply (NADH) were measured by two-photon laser scanning microscopy (2PLSM). Cerebrovascular reactivity (CVR) was evaluated by measuring changes in arteriolar diameters and NADH (tissue oxygen supply) during the hypercapnia test. Transient hypercapnia was induced by a 60-second increase of CO2 concentration in the inhalation mixture from 0% to 10%. Compared to non-diabetic animals, diabetic mice had a significant reduction in the density of functioning capillaries per mm3 (787±52 vs. 449±25), the linear velocity of blood flow (1.2±0.31 vs. 0.54±0.21mm/sec), and the tissue oxygen supply (p<0.05) in both brain and skin. DRP treatment was associated with a 50% increase in all three parameters (p<0.05). According to the hypercapnia test, CVR was impaired in both diabetic groups but more preserved in DRP mice (p<0.05). Our study in a diabetic mouse model has demonstrated the efficacy of hemorheological modulation of blood flow by DRP to achieve increased microcirculatory flows and tissue oxygen supply.

Effect of systemic immunosuppression and BM‐MSC allotransplantation in retinal functionality and RGC population

AbstractPurposeBone marrow mesenchymal stem cell (BM‐MSC) transplant is widely studied in preclinical models because of their potential neuroprotective properties. In the clinic, allotransplants are combined with systemic immunosuppressive treatments to improve transplantation outcomes. We purposed to carry out a comparative functional and histological study to find out the role of immunosuppression after BM‐MSC allotransplantation in retina.Methods20,000 BM‐MSC from donor eGFP‐C57BL/6J mice were intravitreally injected into recipient BALB/c mice. Animals were divided in two groups (n=5/each). One group was immunosuppressed right after the BM‐MSC injection with oral cyclosporine in water (100 mg/ml) and a daily intraperitoneal injection of cortisone (1.6 mg/kg), whereas the other group was kept w/o immunosuppression as control. Electroretinograms were performed before and 5 days and 21 days after the BM‐MSC graft. Animals were perfused, and flat mounted retinas analyzed at 21 days. Brn3a+ retinal ganglion cells (RGC) were automatically quantified, and their spatial distribution analyzed with isodensity maps.ResultsBM‐MSC allotransplant alone causes a significant decrease (40%) of the positive scotopic threshold response (pSTR, RGC function) at 21 days compared to baseline. In the immunosuppressed group, retinal functionality was further impaired as most of the electroretinogram waves (pSTR, rod response, a and b waves, and photopic wave) drastically decreased at 5 days compared to allotransplant alone. This functionality was partially recovered at 21 days. In spite of the functional impairment, the population of RGC was not affected in any group.ConclusionsIn allogeneic BM‐MSC transplants, immunosuppression has a negative effect on retinal function.

Delayed Administration of Nintedanib Ameliorates Fibrosis Progression in CG-Induced Peritoneal Fibrosis Mouse Model

Background: A multiple-target tyrosine kinase inhibitor, nintedanib, which is approved for treatment of interstitial pulmonary disease, has been demonstrated to have anti-fibrotic activity outside of the lungs. We explored its therapeutic effect in a murine model of peritoneal fibrosis. Methods: Daily intraperitoneal injections of chlorhexidine gluconate (CG) induced peritoneal fibrosis in mice. The effects of delayed administration of nintedanib (given at day 21 after CG injection and then given daily for 14 days) were determined by immunohistochemical staining, ELISA, and immunoblot analysis. Results: Delayed administration of nintedanib significantly inhibited peritoneal fibrosis progression as indicated by decreasing deposition and expression of extracellular matrix (ECM) proteins (fibronectin and type I collagen). Treatment with nintedanib also upregulated MMP-2 and reciprocally downregulated TIMP-2, along with reducing expression of α-SMA, β-vimentin, and two transcription factors (Snail and Twist), and retaining E-cadherin expression. Nintedanib also inhibited co-expression of β-vimentin with Snail or Twist as shown by immunofluorescent staining. Moreover, nintedanib decreased the number of CD31-positive blood vessels and CD31 expression in the injured peritoneum. Moreover, delayed application of nintedanib inhibited the expression of several cytokines/chemokines, including monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6, and infiltration of CD68⁺ macrophages to the injured peritoneum. Finally, nintedanib blocked phosphorylation of STAT3, NF-κB, and Smad3 during the development of peritoneal fibrosis. Conclusions: Delayed administration of nintedanib inhibits progression of peritoneal fibrosis and partially reverses established peritoneal fibrosis by attenuating epithelial-mesenchymal transition, inflammation, and angiogenesis, as well as promoting ECM degradation. We conclude that nintedanib has a therapeutic potential to treat peritoneal fibrosis.

A Non-Peptidic MAS1 Agonist AVE0991 Alleviates Hippocampal Synaptic Degeneration in Rats with Chronic Cerebral Hypoperfusion.

Chronic cerebral hypoperfusion (CCH) is a contributing factor for neurodegenerative diseases. As a recently identified heptapeptide of the brain renin-angiotensin system, angiotensin-(1-7) has been revealed to activate its receptor MAS1 and thus ameliorated cognitive impairments in rats with CCH. Since hippocampal synaptic degeneration represents an important pathological basis of cognitive deficits, we hypothesize that activating MAS1-mediated signaling may alleviate CCH-induced synaptic degeneration in the hippocampus. In this study, we tested this hypothesis and uncovered the underlying mechanisms in a rat model of CCH induced by bilateral common carotid artery ligation surgery. At one week after the surgery, rats received a daily intraperitoneal vehicle injection or a non-peptidic MAS1 agonist AVE0991 for 8 weeks. During this procedure, Cerebral Blood Flow (CBF) was recorded. The levels of MAS1, amyloid-β (Aβ), neuroinflammatory cytokines, glial cell markers, and synaptophysin in the hippocampus were assessed at the end of the treatment period. We showed that AVE0991 significantly alleviated hippocampal synaptic degeneration in rats with CCH. This protection might be achieved by facilitating CBF recovery, reducing hippocampal Aβ levels, and suppressing neuroinflammatory responses. These findings indicate that MAS1-mediated signaling may represent a novel therapeutic target for CCH-related neurodegenerative diseases.

Morphofunctional state of the medulla and the fascicular zone of the adrenal cortex in modeling Alzheimer's disease by excessive administration of sodium nitrite

Background. Nitrates and nitrites are found in soil, water, human food, dyes, and medicines. In the literature, both positive and negative effects from the ingestion of nitrates and nitrites into the body are considered. A nitrite model of Alzheimer's type dementia of vascular origin was used in the work. Objective. The aim of the study was to study the morphofunctional state of the adrenal glands of experimental animals with prolonged administration of sodium nitrite. The study was carried out on male rats of the WAG population, which were divided into 3 groups: gr. N-14 - received injections of a water solution of sodium nitrite at a dose of 50 mg / kg of body weight daily for 14 days; gr. N-28 - Rats received similar injections for 28 days. Injections are intraperitoneal. Control rats were injected with 0.9% sodium chloride solution. Results. In animals of the main groups, the formation of a morphofunctional picture of the development of a stress reaction takes place, which was also confirmed morphometrically, and in group N-28 - with signs of incipient decompensation of the medulla and the fascicular zone of the adrenal cortex. Conclusion. When simulating Alzheimer's disease in laboratory rats by the introduction of a water solution of sodium nitrite at a dose of 50 mg / kg of body weight by daily intraperitoneal injection for 14 and 28 days, a picture of their morphofunctional activation is formed in the medulla and fascicular zone of the adrenal cortex. In the group with a 28-day course of administration of sodium nitrite solution, morphofunctional signs of decompensation of the medulla and the fascicular zone of the adrenal glands (especially the medulla) appear, which indicates the development of severe distress in animals and the impossibility of restoring homeostasis.

Voluntary wheel running behaviour as a tool to assess the severity in a mouse pancreatic cancer model.

Laboratory animals frequently undergo routine experimental procedures such as handling, restraining and injections. However, as a known source of stress, these procedures potentially impact study outcome and data quality. In the present study, we, therefore, performed an evidence-based severity assessment of experimental procedures used in a pancreatic cancer model including surgical tumour induction and subsequent chemotherapeutic treatment via repeated intraperitoneal injections. Cancer cell injection into the pancreas was performed during a laparotomy under general anaesthesia. After a four-day recovery phase, mice received either drug treatment (galloflavin and metformin) or the respective vehicle substances via daily intraperitoneal injections. In addition to clinical scoring, an automated home-cage monitoring system was used to assess voluntary wheel running (VWR) behaviour as an indicator of impaired well-being. After surgery, slightly elevated clinical scores and minimal body weight reductions, but significantly decreased VWR behaviour were observed. During therapy, body weight declined in response to chemotherapy, but not after vehicle substance injection, while VWR activity was decreased in both cases. VWR behaviour differed between treatment groups and revealed altered nightly activity patterns. In summary, by monitoring VWR a high impact of repeated injections on the well-being of mice was revealed and substance effects on well-being were distinguishable. However, no differences in tumour growth between treatment groups were observed. This might be due to the severity of the procedures uncovered in this study, as exaggerated stress responses are potentially confounding factors in preclinical studies. Finally, VWR was a more sensitive indicator of impairment than clinical scoring in this model.

Effect of GnRH agonist combined with menstrual blood-derived stem cell transplantation on ovarian function in mice

To investigate the effect of combined treatment with gonadotropin-releasing hormone (GnRH) agonist and menstrual blood-derived stem cell (MenSC) transplantation on ovarian function in mice. We tested the effect of GnRH agonist and MenSC transplantation, either alone or in combination, in a mouse model of premature ovarian failure (POF) induced by daily intraperitoneal injection of 120 mg/kg cyclophosphamide (CTX) for 15 days. Venirelin was administered at the daily dose of 0.1 mg via intravenous infusion, starting at 14 days before modeling; MenSC suspension (2× 106 cells) was injected into the ovarium 1 day after modeling. HE staining was used to observe ovarian pathologies of the mice. ELISA was used to detect serum levels of LH, FSH, AMH and E2. The expression levels of ki-67, Bcl-2, BAX, caspase 9, caspase 3, their cleavages, P-PI3K, PI3K, P-Akt and Akt proteins in ovarian granulosa cells were determined with Western blotting. The mouse models of POF showed obvious ovarian fibrosis with increased atresia follicles and had significantly elevated serum levels of LH and FSH (P < 0.01), decreased serum levels of AMH and E2 (P < 0.01), significantly increased ovarian expression levels of caspase 9, caspase 3, and their cleavages (P < 0.01), and decreased expression levels of Ki-67, Bcl-2, P-PI3K and P-Akt (P < 0.01). The combined treatment with GnRH agonist and MenSC transplantation obviously inhibited the production of ovarian atresia follicles, lowered serum levels of LH and FSH (P < 0.01), and increased the levels of AMH and E2 (P < 0.05 or 0.01) in the mouse models. The combined treatment also inhibited the expressions of BAX and cleaved caspase 9 and caspase 3 proteins (P < 0.05 or 0.01) and enhanced the expressions of Ki-67, Bcl-2, P-PI3K, and p-Akt proteins (P < 0.05 or 0.01) in ovarian granulococcus cells. In mouse models of POF, GnRH agonist combined with MenSC transplantation can reduce the generation of ovarian atresia and improve ovarian reserve by up-regulating PI3K-Akt signaling pathway to achieve ovarian repair and protection.

Oxidative DNA Damage in Tamoxifen Injected THPfl/+ ROSACre/+ Mice

Background and Hypothesis: &#x0D; Tamm-Horsfall protein (THP) is an important regulator of urinary and systemic homeostasis expressed exclusively in the kidney. Complete knockout of THP has been shown to lead to systemic oxidative damage in a mouse model. To develop a more clinically relevant model, we generated a tamoxifen inducible knockout/heterozygote mouse using the Cre/Lox system. We hypothesize that inducing a heterozygous state would increase levels of oxidative damage in mice. &#x0D; Methods: &#x0D; Experimental mice were generated by breeding THPfl/fl mice with ROSACreERT2/CreERT2 mice to develop the inducible heterozygote THPfl/+RosaCreERT2. These mice, along with controls (THPfl/fl) were treated with daily intraperitoneal injections of 75 mg/kg tamoxifen for 5 days. Serum samples were obtained from mice at baseline and 1, 2 and 3 weeks from the first injection, while kidneys were harvested at 1 or 3 weeks. PCR of kidney genomic DNA demonstrated excision of the floxed allele in mice expressing Cre-ERT2. Western Blot analysis of kidney lysates was used to measure kidney THP, while circulating THP was measured by ELISA. Oxidative DNA damage was measured in the kidney and circulation by ELISA. &#x0D; Results: &#x0D; Though kidney THP levels decreased in mice expressing Cre-ERT2, circulating levels of THP remained stable, with evidence of transient increases at 1 or 2 weeks for most animals. Mice expressing Cre-ERT2 had significantly increased oxidative DNA damage within the kidney and there was a trend toward increased oxidative DNA damage in the serum, though larger sample sizes are required to verify this finding. &#x0D; Conclusion: &#x0D; Despite decreased THP in the kidney, mice maintained normal levels of circulating THP. However, higher levels of oxidative damage were found in both the kidney and circulation. Together, these results suggest that THP levels in the serum are tightly controlled and that an acute loss of THP leads to rapid increases in oxidative damage.

Abstract P149: Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer

Abstract Prostate cancer (PC) is the second cause of cancer related deaths in men. Most therapies target the Androgen Receptor (AR) which can prolong survival, but resistance emerges and patients eventually succumb to the disease. This highlights the need for new PC molecular targets. Our group observed in PC patient datasets, the Serine/threoninekinase3 (STK3) gene is frequently amplified and correlates with worse outcomes. This is against the established dogma that STK3 is a tumor suppressor in the Hippo Tumor suppressor pathway that canonically regulates oncogenic transcription factor YAP1. Counter to this role, we report that in PC, STK3 has a pro-tumorigenic role. Our work shows that genetic and chemical inhibition of STK3 in PC cells in vitro significantly slowed proliferation and protasphere growth. The goal of this study was to identify and validate novel STK3 kinase inhibitors with increased potency and specificity. Approach. We screened large assay panels of kinase inhibitors (ChEMBL) for STK3 active molecules, which identified two independent scaffolds. For assessment of STK3 inhibition, we utilized a battery of in cell and biochemical assays including western blot, STK3-NanoBRET assays, radiometric enzymatic assays and a broad KINOMEscan scanMax panels to assess leads and analogs. For phenotypic studies, we utilized Hi-myc ventral prostate (HMVP2) cells to test STK3 inhibition in 3D spheroid growth and spheroid invasion of matrigel by live cell imaging. Pharmacokinetics (PK) and pharmacodynamics (PD) were performed for oral (PO) and intraperitoneal (IP) routes of our top STK3 inhibitor. A subcutaneous HMVP2 allograft model was used to test in vivo efficacy of STK3 inhibition by daily IP injections at 10 mg/kg and 30 mg/kg doses vs. vehicle. Tumor volumes were tracked by caliper measurements and at endpoint, we measured tumor, liver, lung, heart and spleen weights. Results. The two lead scaffolds and a number of analogs showed significant inhibition of STK3 by western blot analysis, in cell STK3-NanoBRET assay and recombinant STK3 radiometric assays. Phenotypic studies in HMVP2 spheres treated with analog STK3 inhibitors showed dose dependent decrease in proliferation and matrigel invasion. From our assays, compound SOB-5-47 emerged as the most potent. PK and PD studies showed that IP injections of SOB-5-47 led to short time in plasma, but pro-longed half-life in the organs such as liver, heart and notably prostate with a terminal half-life clearance of 8.3hrs. Daily injections of SOB-5-47 at 10 and 30 mgs/kg significantly delayed HMVP2 tumor kinetics compared to vehicle control. Tumor weights in SOB-5-47 groups were significantly lower vs control. Importantly, no differences in organ weights or total body weights were observed between groups. Conclusions. Here we report a noncanical role of STK3 in PC and the development of novel STK3 kinase inhibitors that can be used as tools to further examine the role of STK3. Additionally, we show that STK3 can be targeted in vivo to slow PC growth without significant unwanted deleterious effects. Citation Format: Amelia U. Schirmer, Lucy M. Driver, Megan T. Zhao, Carrow I. Wells, Xuan Yang, David H. Drewry, Ivan Spasojevic, Everardo Macias. Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P149.

Metformin induces lactate accumulation and accelerates renal cyst progression in Pkd1-deficient mice.

Metabolic reprogramming is a potential treatment strategy for autosomal dominant polycystic kidney disease (ADPKD). Metformin has been shown to inhibit the early stages of cyst formation in animal models. However, metformin can lead to lactic acidosis in diabetic patients with advanced chronic kidney disease, and its efficacy in ADPKD is still not fully understood. Here, we investigated the effect of metformin in an established hypomorphic mouse model of PKD that presents stable and heritable knockdown of Pkd1. The Pkd1 miRNA transgenic mice of both genders were randomized to receive metformin or saline injections. Metformin was administrated through daily intraperitoneal injection from postnatal day 35 for 4weeks. Unexpectedly, metformin treatment at a concentration of 150mg/kg increased disease severity, including kidney-to-body weight ratio, cystic index and plasma BUN levels, and was associated with increased renal tubular cell proliferation and plasma lactate levels. Functional enrichment analysis for cDNA microarrays from kidney samples revealed significant enrichment of several pro-proliferative pathways including β-catenin, hypoxia-inducible factor-1α, protein kinase Cα and Notch signaling pathways in the metformin-treated mutant mice. The plasma metformin concentrations were still within the recommended therapeutic range for type 2 diabetic patients. Short-term metformin treatment in a second Pkd1 hypomorphic model (Pkd1RC/RC) was however neutral. These results demonstrate that metformin may exacerbate late-stage cyst growth associated with the activation of lactate-related signaling pathways in Pkd1 deficiency. Our findings indicate that using metformin in the later stage of ADPKD might accelerate disease progression and call for the cautious use of metformin in these patients.