C57 Mice Research Articles

ATL I, Acts as a SIRT6 Activator to Alleviate Hepatic Steatosis in Mice via Suppression of NLRP3 Inflammasome Formation.

Accumulating evidence has highlighted that sirtuin-6 (SIRT6) plays an important role in hepatic gluconeogenesis and lipogenesis. We aim to investigate the underlying mechanisms and pharmacological interventions of SIRT6 on hepatic steatosis treatment. Herein, our results showed that atractylenolide I (ATL I) activated the deacetylase activity of SIRT6 to promote peroxisome proliferator-activated receptor alpha (PPARα) transcription and translation, while suppressing nuclear factor NF-kappa-B (NFκB)-induced NACHT, LRR, and PYD domains containing protein 3 (NLRP3) inflammasome formation. Together, these decreased the infiltration of F4/80 and CD11B positive macrophages, accompanied by decreased mRNA expression and serum levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL6), and interleukin-1 beta (IL1β). Additionally, these changes decreased sterol regulatory element-binding protein-1c (SREBP-1c) expression, while restoring carnitine O-palmitoyltransferase 1a (Cpt1a) expression, to decrease the size of adipocytes and adipose deposition, which, in turn, reversed high-fat diet (HFD)-induced liver weight and body weight accumulation in C57 mice. SIRT6 knockout or hepatic SIRT6 knockout in C57 mice largely abolished the effect of ATL I on ameliorating hepatic steatosis. Taken together, our results suggest that ATL I acts as a promising compound that activates SIRT6/PPARα signaling and attenuates the NLRP3 inflammasome to ameliorate hepatic inflammation and steatosis.

Creatine modulates cellular energy metabolism and protects against cancer cachexia-associated muscle wasting.

Cancer cachexia is a multifactorial syndrome defined by progressive loss of body weight with specific depletion of skeletal muscle and adipose tissue. Since there are no FDA-approved drugs that are available, nutritional intervention is recommended as a supporting therapy. Creatine supplementation has an ergogenic effect in various types of sports training, but the regulatory effects of creatine supplementation in cancer cachexia remain unknown. In this study, we investigated the impact of creatine supplementation on cachectic weight loss and muscle loss protection in a tumor-bearing cachectic mouse model, and the underlying molecular mechanism of body weight protection was further assessed. We observed decreased serum creatine levels in patients with cancer cachexia, and the creatine content in skeletal muscle was also significantly decreased in cachectic skeletal muscle in the C26 tumor-bearing mouse model. Creatine supplementation protected against cancer cachexia-associated body weight loss and muscle wasting and induced greater improvements in grip strength. Mechanistically, creatine treatment altered the dysfunction and morphological abnormalities of mitochondria, thus protecting against cachectic muscle wasting by inhibiting the abnormal overactivation of the ubiquitin proteasome system (UPS) and autophagic lysosomal system (ALS). In addition, electron microscopy revealed that creatine supplementation alleviated the observed increase in the percentage of damaged mitochondria in C26 mice, indicating that nutritional intervention with creatine supplementation effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cancer cachexia. These results uncover a previously uncharacterized role for creatine in cachectic muscle wasting by modulating cellular energy metabolism to reduce the level of muscle cell atrophy.

High Morphine Use Disorder Susceptibility Is Predicted by Impaired Learning Ability in Mice.

An obvious reason for substance uses disorders (SUDs) is drug craving and seeking behavior induced by conditioned context, which is an abnormal solid context memory. The relationship between susceptibility to SUD and learning ability remains unclear in humans and animal models. In this study, we found that susceptibility to morphine use disorder (MUD) was negatively correlated with learning ability in conditioned place preference (CPP) in C57 mice. By using behavioral tests, we identified the FVB mouse as learning impaired. In addition, we discovered that learning-relevant proteins, such as the glutamate receptor subunits GluA1, NR1, and NR2A, were decreased in FVB mice. Finally, we assessed the context learning ability of FVB mice using the CPP test and priming. We found that FVB mice had lower learning performance with respect to normal memory but higher performance of morphine-reinstatement memory. Compared to C57 mice, FVB mice are highly sensitive to MUDs. Our results suggest that SUD susceptibility is predicted by impaired learning ability in mice; therefore, learning ability can play a simple and practical role in identifying high-risk SUD groups.

Aβ1‐42 oligomers induce pericyte dysfunction via p75 Neurotrophin Receptor

AbstractBackgroundAmyloid beta (Aβ) aggregates and tau tangles are hallmark pathologies of Alzheimer’s disease (AD). There is growing evidence that vascular dysfunction may contribute to the pathophysiology of AD. Pericyte injury and/or deterioration leading to blood‐brain barrier (BBB) breakdown occur in normal aging and are associated with cognitive impairment in AD patients. Furthermore, there is reduced cerebral blood flow in AD. Because vascular resistance occurs in capillaries, we hypothesize that Aβ1‐42 causes aberrant pericyte contraction and cell death via p75 neurotrophin receptor (p75NTR).Methodp75NTR levels were quantified via Western blotting in mouse vascular brain pericytes treated with either artificial cerebrospinal fluid (aCSF) as a control or 72 nM Aβ1‐42 oligomers for either 20 minutes or 24 hours. Furthermore, LIVE/DEAD assays were performed to quantify pericyte deterioration after exposure to 72 nM Aβ1‐42 for various time points. In mouse models, changes in red blood cell (RBC) flow due to pericyte contraction around capillaries were measured using intravital microscopy in the somatosensory cortex of anesthetized C57 mice and applying either topical aCSF or 72 nM Aβ1‐42.ResultAfter 20 minutes of aCSF or Aβ1‐42 treatment, there is a statistically significant increase in p75NTR levels (n = 3, p = 0.016, two‐tailed t‐test), while there is a statistically significant decrease by 43% in p75NTR levels after 24 hours (n = 6, p = 0.0028, two‐tailed t‐test). After 48 hours of aCSF or Aβ1‐42 treatment, 75.6% of the control pericytes were viable, whereas only 65.3% of the Aβ1‐42 treated pericytes were viable. Preliminary in vivo studies revealed that 72 nM Aβ1‐42 treatment stalled RBC flow.ConclusionPericytes were confirmed to express p75NTR. Aβ1‐42 treatment upregulates p75NTR expression after 20 minutes while downregulating its expression after 24 hours. This suggests that acute increases in p75NTR levels may mediate increased pericyte contraction and play a role in reduced cerebral blood flow during the progression of AD. Because the details of this mechanism are unknown, future in vivo studies will involve pericyte‐specific p75NTR knock‐out mice.

Microglial activation and retinal pigment epithelium alteration in a model of focal LED‐induced phototoxicity. Neuroprotection afforded by bFGF and minocycline

AbstractPurpose: To study the microglial reaction and degeneration of the retinal pigment epithelium (RPE) and its protection with basic fibroblast growth factor (bFGF) administered alone or combined with minocycline (MC) in a model of focal light‐emitting diode (LED)‐induced phototoxicity (LIP) in mice.Methods: In dark‐adapted adult C57 mice (20–25 g), the left eye was dilated and exposed for 45 seconds to a blue LED (400 nm; 500 lux) placed 2 mm perpendicular to the corneal apex (n = 71). Mice were treated with bFGF (0.5 μg) administered alone or in combination with MC (45 mg/kg). bFGF was administered in a single intravitreal injection just after LIP and the MC was daily injected intraperitoneally, starting the day before LIP (n = 10–13 per group). Vehicle or naïve groups were used as controls (n = 6–10 each group). Retinas were immunodetected with anti‐arrestin and anti‐Iba1 antibodies to study cone outer segments (a+OS) and microglial cells (Iba1+cells) and RPEs were immunodetected with anti‐ZO1 to study RPE cells, at 3 and 7 days after LIP. The number of cones and microglial cells was studied within a pre‐fixed area (PFA) of 0.9 mm diameter at the centre of the lesion.Results: Treatment with bFGF alone or in combination with MC was effective in protecting a+OS within the PFA compared to vehicles at 7 days after LIP (p = 0.034 and p = 0.001, respectively). The combined treatment of bFGF and MC was effective in reducing the number of Iba1+cells within the PFA in the outer plexiform layer at 7 days after LIP (p = 0.0003). However, the treatments had no restorative effect on RPE cell survival or morphological integrity at 7 days after LIP (p = 0.6762).Conclusions: Administration of bFGF alone or in combination with MC increased a+OS survival and reduces Iba1+cell activation but does not reduce RPE damage.

Chronic alcohol-induced dysbiosis of the gut microbiota and gut metabolites impairs sperm quality in mice.

Studies have indicated that the ethanol exposure impairs the gut microbiota, At the same time, high levels of alcohol exposure damage sperm in mice. However, whether the gut microbiota is involved in mediating the effects of alcohol on sperm quality remains unclear. This study aimed to assess the effect of chronic alcohol consumption on intestinal microbiota in mice and analyze the potential pathophysiological effect of altered intestinal microbiota on sperm quality. We established a mouse model of chronic alcohol consumption by allowing male C57 mice to freely ingest 10% ethanol for 10 weeks, and collected the fecal microbiota of the male mice in the chronic drinking group (alcohol) and the control group (control) and transplanted the specimens into the transplant groups (the alcohol-fecal microbiota transplantation [FMT] group and the control-FMT group). Sperm quality was significantly decreased in the alcohol-FMT group compared with the control-FMT group. Gut microbiota analysis revealed that the abundance of 11 operational taxonomic units (OTUs) was altered in the alcohol-FMT group. Nontargeted metabolomics identified 105 differentially altered metabolites, which were mainly annotated to amino acids, lipids, glycerophosphoethanolamine, organic oxygenic compounds, organic acids and their derivatives, steroids, and flavonoids. In particular, the oxidative phosphorylation pathway, which is the key to spermatogenesis, was significantly enriched in the alcohol-FMT group. Moreover, compared with the control-FMT group, the alcohol-FMT group presented significantly higher serum endotoxin and inflammatory cytokine levels, with more pronounced T cell and macrophage infiltration in the intestinal lamina propria and elevated levels of testicular inflammatory cytokines. In addition, RNA sequencing showed significant differences in the expression of testis-related genes between the alcohol-FMT group and the control-FMT group. In particular, the expression of genes involved in gamete meiosis, testicular mitochondrial function, and the cell division cycle was significantly reduced in alcohol-FMT mice. In conclusion, these findings indicated that intestinal dysbiosis induced by chronic alcohol consumption may be an important factor contributing to impaired sperm quality. Chronic alcohol consumption induces intestinal dysbiosis, which then leads to metabolic disorders, elevated serum endotoxin and inflammatory cytokine levels, testicular inflammation, abnormal expression of related genes, and ultimately, impaired sperm quality. These findings are potentially useful for the treatment of male infertility.

BMP/Smad Pathway Is Involved in Lithium Carbonate-Induced Neural-Tube Defects in Mice and Neural Stem Cells

Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li2CO3 treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li2CO3 at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li2CO3 or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li2CO3-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li2CO3 exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li2CO3 exposure via inositol deficiency.

Blue light irradiation alleviated dextran sulfate sodium-induced colitis mediated by the Bmal1 pathway in macrophages

Patients with long-term or severe inflammatory bowel disease (IBD) are more likely to develop colorectal cancer. The mortality rate of patients with IBD-related colorectal cancer is also higher than that of patients with common colorectal cancer. This study focused on the effect of photobiomodulation (PBM) on dextran sulfate sodium (DSS)-induced colitis and its underlying mechanism. C57 mice were treated with monochromatic light during DSS-induced colitis. Blue light irradiation clearly alleviated DSS-induced colitis, significantly changed the proportion of the relevant immune cell population in the colon, and improved the inflammatory environment of the colon. Moreover, the rhythm gene Brain And Muscle ARNT-Like 1 (Bmal1) is involved in the progression of colitis, and its decreased expression in colitis mice was reversed by blue light irradiation. These data demonstrated that the protective effect of blue light against colitis is mediated by the Bmal1-mediated pathway in macrophages. This study proposes blue light irradiation with specific light intensities as a potential IBD therapy.

Abstract 13265: Soluble Klotho Captures FGF23 to Mitigate Aortic Valve Inflammocalcification Associated With Chronic Kidney Disease

Calcific aortic valve disease (CAVD) is prevalent in the elderly people, particularly those with chronic kidney disease (CKD). Klotho (KL), an anti-aging protein, is expressed mainly in the kidney and the cardiovascular system as soluble and membrane forms. CKD patients have reduced levels of KL and increased levels of fibroblast growth factor 23 (FGF23), a pro-inflammatory mediator that may interact with soluble KL. We have reported that KL reduces the osteogenic response to high phosphate levels, a condition seen in CKD, in human aortic valve interstitial cells (AVICs). This study tested the hypothesis that soluble KL captures FGF23 to prevent aortic valve inflammocalcification induced by CKD. Methods and Results: We developed a novel CKD-CAVD model in old C57 mice (20 months) on AIN-76A diet and treated with adenine and calcitriol. Histology and immunostaining revealed tubular damage and monocyte/macrophage accumulation in kidney tissue of CKD mice. Aortic valve lesions, thickening and calcification, were evident in CKD mice. Serum FGF23 levels were increased while serum KL levels decreased. In addition, KL expression in the aortic valve was reduced. Recombinant FGF23 (0.04 mg/ml) elevated the inflammosteogenic activity in human AVICs. Treatment with recombinant KL (0.5 μg/ml) markedly decreased FGF23-induced inflammosteogenic activation in AVICs. Systemic analysis revealed that FGF23 upregulates YAP/SMAD3 signaling, and inhibition of YAP and SMAD3 reduced the effect of FGF23 on the inflammosteogenic activity in AVICs. In addition, recombinant KL suppressed the activation of this signaling mechanism in AVICs, and treatment of CKD mice with recombinant KL attenuated aortic valve lesions. However, denaturation of KL protein resulted in a loss of its effect on FGF23-induced AVIC inflammosteogenic activation. Further, incubation of FGF23 with recombinant KL not only resulted in the formation of complexes of these two proteins, but also markedly decreased the pro-inflammosteogenic effect of FGF23 in AVICs. Conclusion: This study demonstrates that CKD induces KL insufficiency and FGF23 accumulation that promote aortic valve lesions. Recombinant KL is capable of binding to FGF23 to suppress its pro-inflammosteogenic effect in the aortic valve.

Effect of Advanced Oxidation Protein Products (AOPPs) and aging on the osteoclast differentiation of Myeloid-Derived Suppressor Cells (MDSCs) and its preliminary mechanism

To verify the osteoclast differentiation ability of MDSCs from mice of different ages and explore the effect of AOPPs on the osteoclast differentiation of bone marrow MDSCs.Bone marrow cells from C57BL/6 (a.k.a C57) mice of different ages were subjected to flow cytometry, and CD11b+Ly6C+Ly6G+ MDSCs were sorted out. After induction of osteoclast differentiation, these cells were subjected to tartrate-resistant acid phosphatase (TRAP) and F-actin. MDSCs from bone marrows of old mice were injected into the tibial medullary cavity of young mice. One week later, the bone marrows were subjected to histological examination, TRAP, and cell count. MDSCs from bone marrows of old mice were sorted for induction of osteoclast differentiation, intervened with reactive oxygen species (ROS) scavenger, inducible nitric oxide synthase (iNOS) inhibitor, and nitric oxide (NO) scavenger, and then subjected to TRAP. 8-weeks-old C57 mice were injected with the same concentrations of either AOPPs or mouse serum albumin (MSA). Four weeks later, MDSCs from bone marrows were sorted and subjected to induction of osteoclast differentiation, followed by IHC staining and TRAP. MDSCs of 8-weeks-old C57 mice were extracted and subjected to in vitro induction of osteoclast differentiation with different concentrations of AOPPs, followed by TRAP training.The number of MDSCs in the bone marrows of old mice was significantly higher than that in young mice. MDSCs from bone marrows of old mice differentiated into large multinucleated TRAP+ osteoclasts, which were significantly different from those in the middle-aged and young mice in terms of cell quantity and morphology. The actin rings formed in the differentiated osteoclasts from MDSCs of bone marrows were densely distributed in the whole field of view, which were significantly denser than those in the middle-aged and young mice. After injection of MDSCs of old mice, the number of TRAP + osteoclasts in the tibial medullary cavity of young mice was significantly increased. NO inhibitor can significantly inhibit the osteoclast differentiation capacity of MDSCs from bone marrows of old mice. In vivo treatment with AOPPs significantly increased the proportion of MDSCs in the bone marrow, which is up to 55.2%. After injection of AOPPs in 8-week-old mice and induction of osteoclast differentiation from the MDSCs, the ratios of CD11b+ and Gr1+ cells were significantly higher than that in the control and MSA groups but was not significantly different from that in the 15-month-old mice. Upon in vitro treatment with different concentrations of AOPPs, the MDSCs did not show any sign of osteoclast differentiation.MDSCs can directly undergo osteoclast differentiation, the capacity of which is stronger in MDSCs of bone marrows of old mice; the NO pathway is a potential mechanism underlying this phenomenon. In vivo but not in vitro AOPPs treatment can induce osteoclast differentiation of MDSCs, indicating there might be other factors in the body that can interact with AOPPs to induce osteoclast differentiation of MDSCs.

Forsythoside B ameliorates diabetic cognitive dysfunction by inhibiting hippocampal neuroinflammation and reducing synaptic dysfunction in ovariectomized mice

Diabetes-associated cognitive impairment (DACI) is a common complication of diabetes, and studies have shown that DACI is more severe in postmenopausal patients with diabetes. Forsythoside B (FTS⋅B) can inhibit inflammation and reduce synaptic dysfunction, which can improve cognitive function. However, it has not been confirmed whether FTS⋅B has a reversing or retarding effect on postmenopausal diabetic encephalopathy. Seven days after bilateral ovariectomy (OVX) or sham surgery, adult female C57 mice (n = 15/group) received intraperitoneal injection of streptozotocin (60 mg/kg/day/L) and citrate buffer for 5 consecutive days to induce diabetes mellitus (DM). Fourteen days later, ovariectomized diabetic mice were given intraperitoneal injection of FTS⋅B (100, 150 mg/kg/day/L) and subcutaneous injection of 17β-estradiol (1 mg/kg) for 8 weeks [OVX + DM + low-FTS⋅B group (L-F), OVX + DM + high-FTS⋅B group (H-F), and OVX + DM + 17β-estradiol (ER)]. In addition, the following control groups were defined: Sham, OVX, DM, and OVX + DM (O + D). Fasting plasma glucose, body weight and blood insulin levels were determined in each group of mice. Next, their cognitive function was tested through behavioral experiments. Hematoxylin & eosin (H&E) and Nissl staining were used to detect the morphological changes in the hippocampus. The aggregation of amyloid beta (Aβ) and the hyperaggregation of p-tau were assessed by immunohistochemistry. Interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), brain-derived neurotrophic factor (BDNF), post-synaptic density-95 (PSD-95), synaptophysin, and synapsin-1 expression in the hippocampus was detected by real-time polymerase chain reaction (RT-PCR) and western blot analysis. FTS⋅B can decrease fasting glucose and blood insulin level. Behavioral results showed that cognitive decline was the most severe in the O + D group, and the ER, L-F, and H-F groups revised the cognitive decline. Compared to the O + D group, more normal morphology, which has obvious nucleoli and clear nuclear membrane, was observed by H&E and Nissl staining in the ER, L-F, and H-F groups. FTS⋅B alleviated DACI by reducing the aggregation of Aβ and the hyperaggregation of p-tau in the hippocampus. Moreover, the protein and mRNA expression showed that FTS⋅B not only inhibited inflammation by decreasing IL-1β, IL-6, and TNF-α but also modulated synaptic plasticity by increasing BDNF, PSD-95, synaptophysin, and synapsin-1. These results suggest that FTS⋅B may be a novel therapeutic target for postmenopausal diabetic encephalopathy treatment.

Effective Echocardiography Parameters to Identify Radiation-Induced Heart Disease in the Mouse Model

<h3>Purpose/Objective(s)</h3> Radiation-induced heart disease (RIHD) may occur after excessive radiotherapy to thoracic cancer. However, the effect of cardiac irradiation on left ventricle (LV) function is still controversial and no previous studies has evaluated the importance of stroke volume (SV) in RIHD. The hypothesis is that diastolic dysfunction with decreased SV could be potentially effective echocardiography parameters to identify RIHD in the mouse model. <h3>Materials/Methods</h3> C57 mice (n=5-10 per group) were treated with a single heart X-ray radiation for 20Gy or 0Gy. Echocardiography was performed to evaluate changes in LV dimensions and function before radiation and after radiation 4, 8 weeks. Elisa kits were used to detect the level of serum BNP and cTnI. HE, Masson's trichrome and Sirius red staining was utilized to assess cardiomyocytes size and myocardial fibrosis. Data are expressed as mean values (means±SEM). Statistical analyses were performed using the two-way repeated measures ANOVA or one-way ANOVA for multiple comparisons and student's t-test for unpaired two groups. In all statistical analyses, significance was defined as P < 0.05. <h3>Results</h3> The Myocardial histopathology indicated a markably increased cardiac fibrosis area and cardiomyocytes size at 8 weeks after radiation. Echocardiography analyses revealed that SV (27.02±1.131 vs. 41.64±2.021ul, P<0.0001), the LV end-diastolic volume (45.64±5.054 vs. 72.24±3.758ul, P=0.0018), and cardiac output (12.58±0.6669 vs. 19.16±0.9942ml/min, P<0.0002) of the 20Gy-mice have significantly decreased than the 0Gy-mice at 8 weeks. The dead 20Gy-mice had lower SV (22.40±0.6110 vs. 30.48±0.1827ul, P=0.0153) as compared to the survival 20Gy-mice. Moreover, the LV diastolic anterior (1.042±0.05123 vs. 0.8456±0.02860mm, P=0.0398) and posterior (0.8745±0.02558 vs. 0.7701±0.02551mm, P=0.0290) wall thickness of 20Gy-mice also had significantly increased than the 0Gy-mice. However, there were no significant difference of the EF (P>0.05), fraction shortening (P>0.05), heart rate (P>0.05), and LV end-systolic volume (P>0.05) between two groups at all the time points. In addition, the result of Elisa kits showed that serum BNP level (427.3±34.05 vs. 279.8±24.84ng/ml, P=0.0127) of 20Gy-mice was significantly higher than the 0Gy-mice at 8 weeks while cTnI was not significantly changed. <h3>Conclusion</h3> Diastolic dysfunction and decreased SV instead of systolic dysfunction occurs firstly in radiation-induced heart disease, which could be effective indexes to detect cardiac toxicity induced by irradiation.

Dapagliflozin attenuates residual cardiac remodeling after surgical ventricular reconstruction in mice with an enlarged heart after myocardial infarction

BackgroundSevere heart failure refractory to conventional therapy requires alternative treatment modalities. Surgical ventricular reconstruction (SVR) has been used to reverse cardiac remodeling in post-myocardial infarction (MI) patients with large left ventricular (LV) aneurysm, however, residual LV remodeling and dysfunction remain postoperatively. It is unclear whether SVR recovers response to drug treatment and whether the sodium-glucose co-transporter 2 inhibitor dapagliflozin (DAPA) reverses residual LV remodeling after SVR. MethodsAdult male C57 mice were subjected to MI or sham surgery. Four-week later, MI mice with LV aneurysm underwent modified SVR or second open-chest sham operation and were randomized to DAPA or vehicle for four-week. Cardiac remodeling, LV function, and the underlying mechanisms were evaluated by echocardiography, invasive LV hemodynamic measurements, mRNA sequencing, and bioinformatics analysis. ResultsSVR significantly decreased LV volume; increased myocardial strain, LV pressure change rates and end-systolic elastance; and decreased heart-to-body weight ratio and myocardial fibrosis. However, significant residual cardiac remodeling remained. DAPA significantly attenuated residual cardiac remodeling and improved LV function in SVR mice but did not have curative effects in non-SVR mice. Of the 1532 genes differentially expressed in SVR and MI mice, 1037 were associated with cardiac metabolism; Src, Crebbp, Fn1, Grb2, and Mapk14 were the top 5 hub genes. Unlike sham surgery, MI upregulated those 5 genes, and treatment with SVR + DAPA normalized their expression. ConclusionsSVR restores therapeutic response in the post-MI heart with large LV aneurysm, and DAPA attenuates residual cardiac remodeling after SVR by normalizing some cardiac metabolism-related hub genes.

Fat transplantation induces dermal adipose regeneration and reverses skin fibrosis through dedifferentiation and redifferentiation of adipocytes

BackgroundLocalized scleroderma causes cosmetic disfigurement, joint contractures, and other functional impairment, but no currently available medications can reverse the resulting skin lesions. Fat grafting is beneficial for reversing skin fibrosis; however, the mechanism by which adipose tissue transplantation contributes to lesion improvement has not been fully clarified. The purpose of our study was to verify the therapeutic effect of fat grafts in reversing skin fibrosis.MethodsInguinal fat pads from AdipoqCreER+;mT/mG mice, which were treated with tamoxifen, were transplanted to the skin lesion in bleomycin-treated wild-type C57 mice. Tdtomato transgenic mice-derived adipocytes, adipose-derived stem cells (ASCs), dedifferentiated adipocytes (DAs) were embedded in matrigel and transplanted beneath the skin lesion of bleomycin-treated wild-type C57 mice. A transwell co‐culture system was used to verify the effect of ASCs, adipocytes or DAs on scleroderma fibroblasts or monocytes.ResultsAdipocytes from the fat grafts could undergo dedifferentiation and redifferentiation for dermal adipose tissue re-accumulation within the skin lesion. Moreover, compared with ASCs and adipocytes, DAs show greater potency of inducing adipogenesis. ASCs and DAs showed comparable effect on inducing angiogenesis and suppressing macrophage infiltration in fibrotic skin. Co-culture assay showed that DAs and ASCs were able to reduce fibrosis-related genes in human scleroderma fibroblasts and drive M2 macrophage polarization.ConclusionOur results indicated that adipocytes would transform into a more functional and dedifferentiated state and reverse dermal fibrosis, by promoting dermal adipose tissue regeneration, improving angiogenesis, suppressing macrophage-mediated inflammation and myofibroblast accumulation.

In Vivo Assessment of the Effects of Mono-Carrier Encapsulated Fucoxanthin Nanoparticles on Type 2 Diabetic C57 Mice and Their Oxidative Stress.

Fucoxanthin (FX) is a carotenoid from a marine origin that has an important role in our health, especially in the regulation and alleviation of type 2 diabetes. Its specific molecular structure makes it very unstable, which greatly affects its delivery in the body. In this study, FX was encapsulated in a mono-carrier using a hydrolyzed zein to form a nanocomplex with a stable structure and chemical properties (FZNP). Its stability was demonstrated by characterization and the efficacy of FX before and after encapsulation in alleviating diabetes in mice, which was evaluated by in vivo experiments. FZNP reduced the level of fasting blood glucose and restored it to normal levels in T2DM mice, which was not caused by a decrease in food intake, and effectively reduced oxidative stress in the organism. Both FX and FZNP repaired the hepatocyte and pancreatic β-cell damage, increased serum SOD and reduced INS values significantly, upregulated PI3K-AKT genes as well as CaMK and GNAs expression in the pancreas. FZNP increased ADPN and GSH-PX values more significantly and it decreased serum HOMA-IR and MDA values, upregulated GLUT2 expression, promoted glucose transport in pancreatic and hepatocytes, regulated glucose metabolism and glycogen synthesis with much superior effects than FX.

S3.5d Identification of fungicidal activity of otilonium bromide by the repurposing approach

S3.5 Environmental exposure - risk for human fungal disease, September 21, 2022, 4:45 PM - 6:15 PM ObjectivesThe development of new antifungal drugs will help to expand the limited antifungal arsenal and curb the emergence of drug resistance. To meet the demand for novel antifungal drugs, we performed a high-throughput screen of an FDA-approved compound library (HY-L022, MCE®) and identified a promising lead antifungal compound, otilonium bromide (OTB).MethodsWe determined antifungal activity of each compound from the library and investigated the antifungal spectrum of OTB using broth microdilution assays (Fig. 1a). We tested the fungicidal effect of OTB by the spot assay and disk diffusion assay. The inhibitory effect of OTB on mycelial growth was identified by observing hyphal growth in hyphal-inducing media containing OTB. The antibiofilm activity of OTB was evaluated by XTT method. We further checked the antifungal activity of OTB in vivo by a murine C. albicans infection model, which was constructed by immunosuppression of mice with cyclophosphamide, inhibiting the intestinal microorganisms by levofloxacin (14 mg/ml), and then gavage with C. albicans cells (1 × 108 cells per mouse). A total of 24 C57 mice were randomly divided into three groups: one group was gavaged with saline, one group with 1 mg/kg fluconazole, and one group with 50 mg/kg OTB. In all 2 h after infection, drugs were given once a day for 4 consecutive days, with 15 days of observation. The death of a mouse was assessed daily and statistically analyzed by Log-rank (Mantel-Cox) test. The toxicity of OTB to human cloned colon adenocarcinoma cells was assessed with MTT method.ResultsWe found that OTB is the most promising lead antifungal molecule among FDA-approved drugs (Fig. 1a). OTB has a broad-spectrum antifungal activity with minimum inhibitory concentration for inhibiting the growth of 50% of strains (MIC50) values ranging from 0.39 to 6.25 μm (Fig. 1b). We found that OTB (≥3.5 μm) has fungicidal effect on C. albicans using spot assay (Fig. 1c). We further found that OTB (25 μg/disk) can form a clear inhibition zone, but not fluconazole (25 μg/disk) (Fig. 1d). Besides, we spotted C. albicans cells from the broth microdilution assays onto YPD solid medium and found that cells treated with OTB (≥1.56 μm) could not recover on YPD solid medium, while cells treated with FLC could (Fig. 1e). It is worth noting that OTB has inhibitory effects on filamentation and biofilm formation at 12.5 μm (Fig. 2a, b), which are key virulence factors of pathogenic fungi. Besides, OTB (50 mg/kg) significantly prolonged the median survival time from 5 days (control group) to 15 days and improved the survival rate from 0% (control group) to 62.5% (P = .0078) (Fig. 2c). Furthermore, we found that OTB exhibited relative low toxicity to mammal cells with IC50 28-57 μm (Fig. 2d), which was much higher than MIC50 against pathogenic fungi.ConclusionOur study highlights that OTB has a broad-spectrum antifungal activity with low MIC50 values and high safety in vitro and potent antifungal efficacy in vivo and will be beneficial to the treatment of devastating invasive fungal infections.

ANGPTL3 is involved in kidney injury in high-fat diet-fed mice by suppressing ACTN4 expression

ObjectiveWe wanted to explore how angiopoietin-like 3 (ANGPTL3) impact hyperlipidemia-induced renal injury.MethodsANGPTL3 knockout mice and wild-type C57 mice were set up in four groups (N = 5) depending on a normal or 60% high-fat diet: wild-type with normal diet (WT), angptl3-/- with normal diet (KO), wild-type + high-fat diet (WT + HF) and angptl3-/- + high-fat diet (KO + HF). The detection time points were the 9th, 13th, 17th and 21st weeks after modeling. Serum lipid and urinary protein levels of mice in each group were detected, and pathological changes in the kidney were analyzed. Moreover, the expression of ANGPTL3, α-actinin-4 (ACTN4), CD2-associated protein (CD2AP) and podocin was tested in the glomerulus by immunohistochemistry (IHC).ResultsIn the WT + HF group, hyperlipidemia and proteinuria could be observed at the 9th week and were gradually aggravated with time. Compared with WT + HF mice, the levels of serum lipids and proteinuria in KO + HF mice were significantly reduced, and the width of podocyte foot processes (FPs) fusion was also markedly improved. The IHC results suggested that in WT + HF mice, the expression of ANGPTL3 was significantly enhanced. After modeling, ACTN4 expression was markedly weakened in the glomeruli of WT + HF mice. Different to WT mice, ACTN4 expression was not observed obviously change in KO + HF mice. Compared with the normal diet group, the expression of podocin showed a decline in WT mice treated with high-fat diet and showed a significant difference from the 17th week. In addition, podocin expression in KO + HF glomeruli was also found to be weak but not significantly different from that in WT + HF glomeruli at the four time points. The expression of CD2AP showed similar results among the four groups.ConclusionANGPTL3 could play a role in the mechanism of hyperlipidemia-associated podocyte injury via ACTN4.

Short-term exposure to JUUL electronic cigarettes can worsen ischemic stroke outcome

BackgroundThe short and long-term health effects of JUUL electronic cigarette (e-Cig) are largely unknown and warrant extensive research. We hypothesized that JUUL exposure could cause cerebrovascular toxicities impacting the progression and outcome of ischemic stroke comparable to tobacco smoke (TS) exposure.MethodsWe exposed male C57 mice to TS/JUUL vapor for 14 days. LCMS/MS was used to measure brain and plasma nicotine and cotinine level. Transient middle cerebral artery occlusion (tMCAO) followed by reperfusion was used to mimic ischemic stroke. Plasma levels of IL-6 and thrombomodulin were assessed by enzyme-linked immunosorbent assay. At the same time, western blotting was used to study blood–brain barrier (BBB) tight junction (TJ) proteins expression and key inflammatory and oxidative stress markers.ResultstMCAO upregulated IL-6 and decreased plasma thrombomodulin levels. Post-ischemic brain injury following tMCAO was significantly worsened by JUUL/TS pre-exposure. TJ proteins expression was also downregulated by JUUL/TS pre-exposure after tMCAO. Like TS, exposure to JUUL downregulated the expression of the antioxidant Nrf2. ICAM-1 was upregulated in mice subjected to tMCAO following pre-exposure to TS or JUUL, with a greater effect of TS than JUUL.ConclusionsThese results suggest that JUUL exposure could negatively impact the cerebrovascular system, although to a lesser extent than TS exposure.

Nitrative inactivation of thioredoxin-1 loses its protective effect in bleomycin-induced pulmonary fibrosis

Pulmonary fibrosis is common in the development of inflammatory lung diseases with no effective clinical drug treatment currently. As an essential redox enzyme, thioredoxin (Trx) has been reported to be involved in pulmonary fibrosis, but the mechanism is to be revealed. Therefore, in bleomycin-indued pulmonary fibrosis model in C57 mice, Trx activity and nitrated Trx were examined.,p38-MAPK apoptosis pathway was determined in lung tissues. Additionally, before BLM administration, C57/BL6 mice were treated with aminoguanidine (AG, a peroxynitrite scavenger), recombinant human Trx-1 (rhTrx-1), or SIN-1 (a peroxynitrite donor) nitrated Trx-1 (N-Trx-1). In bleomycin (BLM)-induced pulmonary fibrosis model in C57/BL6 mice, we observed that nitrated Trx increased, while its activity decreased, with the increase of alveolar epithelial cells (AECs)apoptosis by p38-MAPK pathway. We demonstrated that AG or rhTrx-1, but not N-Trx-1 significantly reduced pulmonary fibrosis. Taken together, the results above revealed that blockade of Trx-1 nitration, or supplementation of exogenous rhTrx-1, might represent novel therapies to attenuate pulmonary fibrosis in idiopathic pulmonary fibrosis patients.

Evaluation of Hypoglycemic Activity and Sub-Acute Toxicity of the Novel Biochanin A-Chromium(III) Complex.

The novel biochanin A–chromium(III) complex was synthesized by chelating chromium with biochanin A (BCA). The structure of the complex was determined and the complex ([CrBCA3]) was composed of chromium(III) and three ligands, and the chromium content was 55 μg/mg. The hypoglycemic activity of the complex was studied in db/db mice and C57 mice. The sub-acute toxicity test of the complex was carried out by the maximum limit method in KM mice. The hypoglycemic activity showed that the complex could reduce the weight of db/db mice and lower the fasting blood glucose and random blood glucose levels. The complex also improved the organ index, oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) results of db/db mice, and some of the indicators were similar to those of the positive control group after treatment with the complex. The histopathology study showed significant improvements in the liver, kidney, pancreas and skeletal muscle compared with the diabetes model group. The complex also showed a significant improvement in serum biochemical indices and antioxidant enzyme activities, as well as glycogen levels. The sub-acute toxicity study showed that the complex did not cause death or any dangerous symptoms during the study. In addition, the sub-acute toxicity study showed that the complex had no significant effect on the serum biochemical indices, antioxidant capacity and organs of normal mice. This study showed that [CrBCA3] had good hypoglycemic activity in vivo and had no sub-acute toxicity. This work provides an important reference for the development of functional hypoglycemic foods or drugs.