Effects of amyloid ß (Aß)42 and Gasdermin D on the progression of Alzheimer's disease in vitro and in vivo through the regulation of astrocyte pyroptosis.

PURPOSE: The study aimed to investigate whether astrocyte pyroptosis, and the subsequent neuroinflammatory response that exerts amyloid ß (Aß) neurotoxic effects, has an effect on endothelial cells, along with the underlying mechanisms. METHODS: In vivo, 5 µL of disease venom was injected into the lateral ventricle of APP/PS1 mice for treatment. Pyroptosis was induced by treating astrocytes with Aß42 in vitro. Small interfering RNA (siRNA) was used to silence caspase-1 and Gasdermin D (GSDMD) mRNA expression. Cell viability was determined using a CCK-8 detection kit. Scanning electron microscopy (SEM), Annexin V/propidium iodide (PI) double staining, RT-qPCR, immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. The degree of pathological damage to the brain and aortic tissue was assessed by hematoxylin-eosin staining and immunohistochemistry. RESULTS: Aß42 induced astrocyte pyroptosis dependent on the GSDMD/Gasdermin E (GSDME)/Caspase 11/NLRP3 pathway, releasing large amounts of inflammatory factors, such as TNF-α, IL-1α, IL-1ß, and IL-18. Astrocyte pyroptosis caused endothelial cell dysfunction and release of large amounts of vasoconstrictors (ET and vWF). Knockdown of GSDMD reduced astrocyte pyroptosis in the cerebral cortex and hippocampal tissue, decreased the release of inflammatory factors IL-1 ß and IL-18, reduced Aß deposition and tau protein, increased the release of peripheral vasodilator substances (eNOS), and decreased the release of vasoconstrictor substances (ET, vWF), thereby reducing brain tissue damage and vascular injury in APP/PS1 mice. CONCLUSION: Aß42 induced astrocyte pyroptosis, while GSDMD knockout inhibited astrocyte pyroptosis, reduced the release of inflammatory factors, and alleviated brain tissue damage and vascular damage in APP/PS1 mice. Therefore, GSDMD is a novel therapeutic target for Alzheimer's disease. PURPOSE: The study aimed to investigate whether astrocyte pyroptosis, and the subsequent neuroinflammatory response that exerts amyloid ß (Aß) neurotoxic effects, has an effect on endothelial cells, along with the underlying mechanisms. METHODS: In vivo, 5 µL of disease venom was injected into the lateral ventricle of APP/PS1 mice for treatment. Pyroptosis was induced by treating astrocytes with Aß42 in vitro. Small interfering RNA (siRNA) was used to silence caspase-1 and Gasdermin D (GSDMD) mRNA expression. Cell viability was determined using a CCK-8 detection kit. Scanning electron microscopy (SEM), Annexin V/propidium iodide (PI) double staining, RT-qPCR, immunofluorescence, western blotting, and enzyme-linked immunosorbent assay (ELISA) were used to detect cell pyroptosis. The degree of pathological damage to the brain and aortic tissue was assessed by hematoxylin-eosin staining and immunohistochemistry. RESULTS: Aß42 induced astrocyte pyroptosis dependent on the GSDMD/Gasdermin E (GSDME)/Caspase 11/NLRP3 pathway, releasing large amounts of inflammatory factors, such as TNF-α, IL-1α, IL-1ß, and IL-18. Astrocyte pyroptosis caused endothelial cell dysfunction and release of large amounts of vasoconstrictors (ET and vWF). Knockdown of GSDMD reduced astrocyte pyroptosis in the cerebral cortex and hippocampal tissue, decreased the release of inflammatory factors IL-1 ß and IL-18, reduced Aß deposition and tau protein, increased the release of peripheral vasodilator substances (eNOS), and decreased the release of vasoconstrictor substances (ET, vWF), thereby reducing brain tissue damage and vascular injury in APP/PS1 mice. CONCLUSION: Aß42 induced astrocyte pyroptosis, while GSDMD knockout inhibited astrocyte pyroptosis, reduced the release of inflammatory factors, and alleviated brain tissue damage and vascular damage in APP/PS1 mice. Therefore, GSDMD is a novel therapeutic target for Alzheimer's disease.

MiR-590-3p Promotes the Phenotypic Switching of Vascular Smooth Muscle Cells by Targeting Lysyl Oxidase.

ABSTRACT: We investigated the clinical characteristics of patients with acute aortic dissection (AAD) and miR-590-3p levels in serum, tissue, and vascular smooth muscle cells. The effect of miR-590-3p on the vascular smooth muscle cell phenotype was assessed, and the regulation of lysyl oxidase by miR-5903p was determined. C57BL/6 mice were used to investigate the incidence of AAD and effects of miR-5903p on AAD. The miR-590-3p levels were measured in the aortae of mice, and hematoxylin and eosin staining and Masson staining were performed to identify the morphological features of the aorta. Comparative analysis revealed significant differences in clinical characteristics between patients with AAD and healthy control subjects, with most patients with AAD exhibiting concomitant hypertension and nearly 50% having atherosclerosis. Lysyl oxidase was a direct target of miR-590-3p. Lysyl oxidase overexpression inhibited switching of the vascular smooth muscle cell phenotype from contractile to synthetic, but miR-590-3p overexpression significantly reversed this change. In the mouse model, miR-590-3p upregulation increased the incidence of AAD to 93.3%, and its incidence decreased to 13.3% after miR-590-3p inhibition. Hematoxylin and eosin and Masson staining revealed that the miR-590-3p agomiR group had a greater loss of the contractile phenotype in the dissected aortic wall and an increased number of muscle fibers in the aortic wall, which contributed to thickening of the aortic wall and the formation of a false lumen in aortic dissection. miR-590-3p might be pivotal in the pathogenesis of AAD. Thus, targeting miR-590-3p or its downstream pathways could represent a therapeutic approach for AAD.

Hematoxylin and Eosin staining of PhenoCycler® Fusion flow cell slides.

Multiplexed Imaging technologies are powerful techniques that enable ultrahigh-plex spatial phenotyping of whole tissue sections at single cell spatial resolution. Co-Detection by Indexing (CODEX) multiplexing can detect up to 100 proteins using cyclic detection of DNA conjugated antibodies applied to tissue sections. However, it is necessary to correlate multiplexed fluorescent (mIF) spatial images with Hematoxylin and Eosin (H&E) stained sections post analysis. To effectively correlate mIF spatial images with H&E morphology, an (H&E) staining protocol was developed that is directly applied to the CODEX Fusion flow-cell slide after analysis allowing for direct H&E correlation and annotation with mIF images.

Salidroside Inhibits Ganglion Cell Apoptosis by Suppressing the Müller Cell Inflammatory Response in Diabetic Retinopathy.

PURPOSE: This study aimed to investigate the role of salidroside (SAL) in the cellular communication between Müller cells and retinal ganglion cells in diabetic mice. METHODS: The diabetes mellitus (DM) animal models were established by the intraperitoneal injection of streptozotocin and treatment with SAL via gavage or by the injection of IL-22BP into the vitreous cavity. Immunohistochemistry was used to measure the expression of the glial fibrillary acidic protein in Müller cells. The expression of IL-22 and IL-22Rα1 in retinal tissues was assessed by immunofluorescence. Western blotting was used to measure the expression of inflammatory and apoptosis-related proteins. Hematoxylin-eosin staining, TUNEL staining, and flow cytometry were used to analyze the apoptosis of retinal ganglion cells. The effect of cellular interactions was explored by Transwell assays. RESULTS: Western blotting showed that glial fibrillary acidic protein, IL-22 protein expression was significantly upregulated in the DM animal models compared with the control mice. Immunofluorescence showed that IL-22 was highly expressed in Müller cells and IL-22Rα1 was expressed in ganglion cells in the retina of DM mice. Hematoxylin-eosin and TUNEL staining results showed an increase in the number of ganglion cells apoptotic in DM. However, SAL reversed these phenomena. Meanwhile, after coculture with Müller cells, Western blotting suggested that ganglion cells secreted p-STAT3, and c-caspase3 protein expression was increased. More interestingly, the treatment of IL-22BP and SAL inhibited the expression of the p-STAT3 and c-caspase3 proteins. Flow cytometry indicates that compared with the control group, the apoptosis rate of ganglion cells was increased in the high glucose group, while the apoptosis rate of cells in the recombinant IL-22 protein group was significantly increased, while the SAL inhibited ganglion cells apoptosis. CONCLUSION: SAL inhibits the apoptosis of retinal ganglion cells via the IL-22/STAT3 pathway in Müller cells.

Myricitrin promotes osteogenesis and prevents ovariectomy bone mass loss via the PI3K/AKT signalling pathway.

This study aimed to explore the effect of myricitrin on osteoblast differentiation in mice immortalised bone marrow mesenchymal stem cells (imBMSCs). Additionally, ovariectomy (OVX) mice were employed to examine the effect of myricitrin on bone trabecular loss in vivo. The effect of myricitrin on the proliferation of imBMSCs was evaluated using a cell counting kit-8 assay. Alizarin red staining, alkaline phosphatase staining were performed to elucidate osteogenesis. Furthermore, qRT-PCR and western blot determined the expression of osteo-specific genes and proteins. To screen for candidate targets, mRNA transcriptome genes were sequenced using bioinformatics analyses. Western blot and molecular docking analysis were used to examine target signalling markers. Moreover, rescue experiments were used to confirm the effect of myricitrin on the osteogenic differentiation of imBMSCs. OVX mice were also used to estimate the delay capability of myricitrin on bone trabecular loss in vivo using western blot, micro-CT, tartaric acid phosphatase (Trap) staining, haematoxylin and eosin staining, Masson staining and immunochemistry. In vitro, myricitrin significantly enhanced osteo-specific genes and protein expression and calcium deposition. Moreover, mRNA transcriptome gene sequencing and molecular docking analysis revealed that this enhancement was accompanied by an upregulation of the PI3K/AKT signalling pathway. Furthermore, copanlisib, a PI3K inhibitor, partially reversed the osteogenesis promotion induced by myricitrin. In vivo, western blot, micro-CT, hematoxylin and eosin staining, Masson staining, Trap staining and immunochemistry revealed that bone trabecular loss rate was significantly alleviated in the myricitrin low- and high-dose groups, with an increased expression of osteopontin, osteoprotegerin, p-PI3K and p-AKT compared to the OVX group. Myricitrin enhances imBMSC osteoblast differentiation and attenuate bone mass loss partly through the upregulation of the PI3K/AKT signalling pathway. Thus, myricitrin has therapeutic potential as an antiosteoporosis drug.

Selenium deficiency causes oxidative stress and activates inflammation, apoptosis, and necroptosis in the intestine of weaned calves.

Selenium performs a variety of biological functions in organisms, including antioxidant and anti-inflammatory effects. This study investigated how selenium deficiency affects weaned calves' intestines. According to Inductively coupled plasma mass spectrometry (ICP-MS) analysis of intestinal selenium concentrations in calves, the Se-D group had a significantly lower concentration of selenium. Hematoxylin-eosin staining showed that the intestinal epithelial cells were detached, the goblet cells were lost, and the intestinal villi were fragmented and loosely arranged in the Se-D group, along with hyperemia and inflammatory infiltration. Of the 22 selenoprotein genes, 9 were downregulated in response to selenium deficiency in Reverse transcription-PCR (RT-PCR), whereas 6 genes were upregulated. In the Se-D group, oxidative stress was detected by measuring redox levels in the intestines. Furthermore, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, RT-PCR, and Western blotting (WB) results indicated that both intrinsic and extrinsic apoptosis pathways are activated in the intestine during selenium deficiency. Selenium deficiency also induced necroptosis in the intestine through upregulation of MLKL, RIPK1, and RIPK3 mRNA levels. In addition, according to hematoxylin-eosin staining and ELISA, selenium-deficient calves had severe inflammation in their intestines. As a result of RT-PCR and WB analyses, we found that selenium deficiency was associated with nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Our study suggested that weaned calves' intestines are affected by selenium deficiency, which causes oxidative stress, inflammation, apoptosis, and necroptosis.

Danhong injection alleviates cerebral ischemia-reperfusion injury by inhibiting autophagy through miRNA-132-3p/ATG12 signal axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danhong injection (DHI) is a renowned traditional Chinese medicine often used clinically to treat cardiovascular and cerebrovascular diseases. Studies have shown that DHI can significantly alter microRNA (miRNA) expression in the brain tissue. Therefore, exploring specific miRNAs' regulatory mechanisms during treatment with DHI is essential. AIM OF THE STUDY: To investigate DHI's regulatory mechanism on cerebral autophagy in rats with cerebral ischemia-reperfusion injury (CIRI). MATERIAL AND METHODS: Rats were randomly divided into the sham, middle cerebral artery occlusion (MCAO) model, and DHI-treatment groups. The extent of brain damage was evaluated using triphenyl tetrazolium chloride and hematoxylin-eosin staining. Hippocampal cell autophagy was observed using transmission electron microscopy. Autophagy-related proteins were analyzed using western blotting. Differentially expressed miRNAs were screened using high-throughput and real-time quantitative reverse transcription PCR. The relationship between miR-132-3p and ATG12 was confirmed using a dual-luciferase assay. The miR-132-3p mimics and inhibitors were transfected into PC12 cells subjected to oxygen-glucose deprivation (OGD) in vitro and MCAO model rats in vivo. RESULTS: DHI significantly altered the miRNA expression profile in rat brain tissues. The pathological changes in the brain tissues were improved, and the autophagic hippocampal cell vehicles were significantly reduced after DHI treatment. miRNA-132-3p, one of the miRNAs with a significantly different expression, was screened. Kyoto Encyclopedia of Genes and Genomes signal pathway analysis showed that its target genes were closely related to autophagy. Western blotting revealed that the p-PI3K, p-AKT, and mTOR expression increased significantly; AMPK, ULK1, ATG12, ATG16L1, and LC3II/I were downregulated in the DHI group. Dual-luciferase reporter gene experiments showed that miRNA-132-3p could target the ATG12 3'-UTR region directly. In vitro, miRNA-132-3p had a protective effect on OGD/R-induced oxidative stress injury in PC12 cells, improving cell viability, and affecting the expression of autophagy pathway-related proteins. In vivo transfection experiments showed that miR-132-3p could regulate ATG12 expression in CIRI rats' lateral brain tissue, affecting the autophagy signaling pathway. miR-132-3p overexpression reduces CIRI-induced autophagy and protects neurons. CONCLUSION: This study showed that DHI inhibits neuronal autophagy after cerebral ischemia-reperfusion. This may have resulted from miR-132-3p targeting ATG12 and regulating the autophagy signaling pathway protein expression.

Saffron essential oil ameliorates CUMS-induced depression-like behavior in mice via the MAPK-CREB1-BDNF signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Saffron, the dried stigma of Crocus sativus L., has a long history of use in the treatment of depression in traditional Chinese medicine and Islamic medicine. The unique aroma of saffron, primarily derived from its volatile oil, has been widely used by folk to mitigate anxiety and depression via sniffing because the aroma of saffron has a pleasant and invigorating effect. AIM OF THE STUDY: This study aimed to investigate the antidepressant effect and the underlying mechanism of saffron essential oil (SEO) in mice exposed to chronic unpredictable mild stress (CUMS). MATERIALS AND METHODS: In this study, compounds of SEO were identified using gas chromatography-mass spectrometry analysis, while network pharmacology was used to predict potential active compounds, antidepressant targets, and related signaling pathways of SEO. The CUMS depression model was further used to explore the therapeutic effect and possible mechanism of SEO. During the modeling period, mice were regularly administered fluoxetine (3.6 mg/kg, i.g.) or diluted SEO (2%, 4%, and 6% SEO, inhalation). The antidepressant and neuroprotective effects of SEO were evaluated by behavior tests (the open field test, the sucrose preference test, the tail suspension test, and the forced swimming test), hematoxylin-eosin staining, and Nissl staining. The enzyme-linked immunosorbent assay kits were used to measure dopamine (DA), 5-serotonin (5-HT), brain-derived neurotrophic factor (BDNF), and γ-aminobutyric acid (GABA) levels in serum. The relative abundance of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B in the hippocampus was determined using western blot (WB). RESULTS: According to the network pharmacology analysis, seven active SEO compounds mediated 113 targets related to depression treatment, most of which were enriched in the 5-HT synapse, calcium signaling pathway, and cAMP signaling pathway. In vivo experiments indicated that fluoxetine and SEO improved depression-like behaviors in depressed mice. The levels of 5-HT, DA, BDNF, and GABA in serum increased significantly. Histopathological examinations revealed that fluoxetine and SEO ameliorated neuronal damage in the hippocampus. WB analysis showed that the relative expressions of Raf1, MEK1, P-ERK1/2/ERK1/2, P-CREB1/CREB1, BDNF, and P-Trk B/Trk B were significantly higher in the fluoxetine and SEO groups than in the CUMS group. CONCLUSION: Overall, these findings suggest that SEO significantly alleviates the depressive symptoms in CUMS exposed mice and partially restores hippocampal neuronal damage. Meanwhile, the best efficacy was observed in 4% SEO. Furthermore, the antidepressant mechanism of SEO is primarily dependent on the regulation of the MAPK-CREB1-BDNF signaling pathway.

Fufang-zhenzhu-tiaozhi formula protects islet against injury and promotes ß cell regeneration in diabetic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-zhenzhu-tiaozhi formula (FTZ) is a patented preparation of traditional Chinese medicine that has been used to treat hyperglycemia and hyperlipidemia in the clinic for almost 10 years. Our previous study had demonstrated that FTZ can protect islet ß cell injury in vitro. However, the efficacy of FTZ on ß cell regeneration in vivo and the involved anti-diabetic mechanism remains unknown. AIM OF THE STUDY: We aim to investigate the effects of FTZ as a good remedy for islet protection and ß cell regeneration, and to reveal the underlying mechanism. MATERIALS AND METHODS: C57BL/6 mice were fed with high-fat diet for 3 weeks and then intraperitoneally injected with streptozotocin (90 mg/kg/d × 1 d) to establish type 2 diabetes (T2D) models. Mice in each group were divided into three batches that sacrificed after 3, 7 and 28 days of FTZ administration. Body weight, blood glucose, and oral glucose tolerance test were measured at indicated time points. Fasting insulin was determined by enzyme-linked immunosorbent assay (ELISA) kit. Neonatal ß cell was assessed by insulin & PCNA double immunofluorescence staining, and the underlying mechanisms related to ß cell regeneration were further performed by hematoxylin-eosin staining, insulin & glucagon double immunofluorescence staining and Western blot. RESULTS: FTZ and metformin can significantly help with the symptoms of DM, such as alleviating weight loss, reducing blood glucose, improving the level of insulin in vivo, and relieving insulin resistance, suggesting FTZ and metformin treatment maintained the normal morphological function of islet. Notably, ß cell regeneration, which is indicated by insulin and PCNA double-positive cells, was promoted by FTZ, whereas few neonatal ß cells were observed in metformin group. Hematoxylin-eosin staining, and its quantification results showed that FTZ effectively prevented the invasion of inflammatory cells into the islets in diabetic mice. Most ß cells in the islets of diabetic model mice were devoid, and the islets were almost all α cells, while the diabetic mice administered FTZ could still maintain about half of the ß cells in the islet. Furthermore, FTZ upregulated the expression of critical transcription factors during ß cell development and maturation (such as PDX-1, MAFA and NGN3) in diabetic mice. CONCLUSIONS: FTZ can alleviate diabetes symptoms and promote ß cell regeneration in diabetic mice. Moreover, FTZ promotes ß cell regeneration by preserving islet (resisting inflammatory cells invading islets), maintaining the number of ß cells in islets, and increasing the expression of PDX-1, MAFA and NGN3.

The impact of IGF-1 on alveolar bone remodeling and BMP-2 expression in orthodontic tooth movement in diabetic rats.

BACKGROUND: Orthodontic tooth movement is linked to alveolar bone reconstruction. OBJECTIVES: As a regulator of cell proliferation, insulin-like growth factor 1 (IGF-1) plays an important role in osteoporotic fracture healing. This study aims to investigate the effect of IGF-1 on alveolar bone remodeling in diabetic rats. MATERIAL AND METHODS: Sprague Dawley (SD) rats were randomly divided into 3 groups, including a control group, a model group established with streptozotocin (STZ) injection to prepare the diabetic rats (type 1 diabetes), and an IGF-1 group of diabetic rats receiving daily intraperitoneal injections of 1.0 mg/kg IGF-1. Nickel-titanium coil springs were used to pull the first molar forward to establish the model. The maxillary first to third molars and the surrounding alveolar bone were collected to measure tooth movement distance. Hematoxylin and eosin (H&E) staining was applied to detect the pathological changes in the periodontal tissue. Real-time polymerase chain reaction (PCR) and western blot were adopted to measure bone morphogenetic protein 2 (BMP-2) mRNA and protein expression. Enzyme-linked immunosorbent assays (ELISAs) were used to measure interleukin-1α (IL-1α) levels in the serum. RESULTS: The tooth movement distance was significantly decreased, BMP-2 expression was downregulated, and IL-lα levels were enhanced in the model group compared to the control group (p < 0.05). However, the tooth movement distance was increased, BMP-2 expression was increased, and IL-lα levels were reduced in the IGF-1 group compared to the model group (p < 0.05). Hematoxylin and eosin staining showed that alveolar bone destruction was attenuated in the IGF-1 group, while the new bone was not active in the model group. CONCLUSIONS: Diabetes can damage alveolar bone remodeling in orthodontic tooth movement. The IGF-1 promotes alveolar bone remodeling by inhibiting inflammation and upregulating BMP-2 expression.

Xianglian Pill attenuates ulcerative colitis through TLR4/MyD88/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xianglian Pill (XLP) is a classical Chinese medicine prescription applied for controlling ulcerative colitis (UC). Whereas, the underlying mechanism remains unclear. AIM OF THE STUDY: The present work was aimed to investigate the mechanism of XLP in dextran sulfate sodium (DSS)-induced UC via the Toll Like Receptor 4 (TLR4)/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-B (NF-κB) signaling in mice. MATERIALS AND METHODS: The major components of XLP were detected by high-performance liquid chromatography-diode array detection (HPLC-DAD). The ulcerative colitis model was induced by DSS in mice. 5-Amino Salicylic Acid (5-ASA) group and XLP group were intragastrically treated. Disease activity index (DAI) and colon length were monitored and hematoxylin-eosin (HE) staining was conducted. Gasdermin D (GSDMD)-N and TLR4 expressions in colon tissues were visualized by immunofluorescence. TLR4 mRNA was measured by Real Time Quantitative PCR (RT-qPCR). The expressions of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), active-caspase-1, GSDMD-N, TLR4, MYD88, NF-κB, p-NF-κB, and the ubiquitination of TLR4 in colon tissues were detected by Western blot. Myeloperoxidase (MPO) enzyme activity was examined and serum inflammatory factors Interleukin (IL)-1ß, IL-6, Tumor Necrosis Factor-α (TNF-α), and IL-18 were determined by Enzyme-linked Immunosorbent Assay (ELISA). TLR4-/- mice were applied for verifying the mechanism of XLP attenuated DSS symptoms. RESULTS: The XLP treatment extended colon length, reduced DAI, and attenuated histopathological alteration in DSS-induced mice. XLP administration suppressed MPO activity and reduced the content of IL-1ß, IL-6, TNF-α and IL-18 in serum. XLP also inhibited the expression levels of GSDMD-N, TLR4, NLRP3, active-caspase-1, MyD88, p-NF-κB/NF-κB in colon tissues of DSS-induced mice. TLR4-/- mice proved that TLR4 was involved in XLP-mediated beneficial effect on DSS-induced ulcerative colitis. CONCLUSIONS: XLP might treat ulcerative colitis by regulating the TLR4/MyD88/NF-κB signaling pathway.

Swimming exercise and nano-l-arginine supplementation improve oxidative capacity and some autophagy-related genes in the soleus muscle of aging rats.

The present research aims to evaluate the effect of swimming exercise and chitosan-coated l-arginine on mitochondrial oxidation, BCL2 Interacting Protein 3 (Bnip3), NIP-like protein × (Nix), B-cell lymphoma-extra-large (Bcl-xL) and autophagy-related protein light chain 3(LC3) expression in soleus muscle of aging rats. In this experimental research, 25 male Wistar rats were assigned into five groups randomly: young, old, old + Nano l-arginine (Nano L-a), old + exercise (Ex), and old + Nano l-arginine (Nano L-a) + exercise (Ex) (n = 5 in each). They performed a swimming exercise program five days a week for six weeks. To determine the relative strength for rats before and after performing these interventions, the 1repetition maximum (1RM) test was done as a pre and post-test. The exercise program started with 20 min and after four sessions, gradually increased to 60 min and this time was maintained until the completion of the training period. l-arginine coated with chitosan nanoparticles was given to the rats in the l-arginine-supplemented group via gavage at a dosage of 500 mg/kg/day, five days a week, for six weeks. Additionally, the rats in all groups were fed a normal diet (2.87 kcal/g and 15 % energy from fat). Upon the completion of the protocol implementation, the rats were sacrificed and the soleus muscle was fixed and frozen to determine hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), gene expression analysis, levels of reactive oxygen species (ROS), and total antioxidant capacity (TAC). The results from the present research indicated that swimming exercise and Nano l-arginine improve the strength and histology of muscle tissue in old rats (p < 0.05). Aging significantly increased the expression of Nix and Bnip3 (p < 0.05) and reduced the Bcl-xL gene expression (p < 0.05). The expression of LC3 protein also increased with aging (p < 0.05). Therapeutic interventions, such as combined treatment (old + Nano L-a + Ex) for old animals, reduced the amount of this protein in soleus muscle (p < 0.05). The ROS values also showed a significant reduction only in the old + Nano L-a + Ex group compared to the old group. Moreover, TAC values show a significant decrease in the old and old + Ex groups in comparison to the young group. The use of arginine supplement, especially in nano form, along with swimming exercise seems to reduce the oxidative damage to the elderly muscle tissue, which has a positive effect on the structure and function of the soleus muscle. Since these interventions only had a significant effect on LC3 protein, further studies with more diverse measurement methods for autophagy are suggested.

Healing Potential of Propolis Extract-Passiflora edulis Seed Oil Emulgel Against Excisional Wound: Biochemical, Histopathological, and Cytokines Level Evidence.

Propolis is rich in natural bioactive compounds, and considering its importance for many skin therapies, emulgel was prepared. This study examines how a propolis extract (PE) and Passiflora edulis seed (PS) oil emulgel affect rat deep skin wound healing. Based on preset criteria of maximum drug content and optimum drug permeation through the stratum corneum along with drug retention in the skin layers, an optimized emulgel formula based on Box-Behnken factorial design was prepared and used for subsequent in vitro and in vivo evaluations. In vivo wound-healing activities of emulgel and control treatments were investigated in a rat model. The optimized emulgel formula exhibited superior healing activity compared with plain PE suspension-treated rats on day 14 of wounding. Histopathological investigations of hematoxylin and eosin and Masson's Trichrome-stained skin sections supported this effect. Emulgel promotes cutaneous wound healing through a variety of mechanisms, including anti-inflammatory through modulation of cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 production, and promotion of collagen fiber formation, all of which contribute to tissue remodeling. Furthermore, when compared with propolis suspension, emulgel showed significant antioxidant and anti-inflammatory effects. Emulgel significantly increased the skin's hydroxyproline level, antioxidant potential, wound contraction, increased penetration, and localized propolis deposition across the skin. Incorporation of PS oil into the emulgel accelerates the tissue regeneration process. The findings suggest that 5% propolis emulgel could be used as an alternative to treat wounds.

Histometric and morphological damage caused by Serratia marcescens to the tick Rhipicephalus microplus (Acari: Ixodidae).

Rhipicephalus microplus tick is the ectoparasite causing the greatest economic losses in the livestock industry. Multi-resistance in ticks is increasing, generating the inefficiency of traditional ixodicides, for which biological control has been proposed as an alternative. In this work, we analyze the histomorphological damage caused by the bacterial strain EC-35 on Rhipicephalus microplus. The ixodicidal effect of EC-35 total protein was evaluated on larval or adult ticks comparing with the commercial ixodicide coumaphos 0.02% as a control. Female ticks were processed using the paraffin-embedding technique and stained with hematoxylin-eosin. Also, the pathogenicity of EC-35 was evaluated by capillary feeding and coelom inoculation tests. The identification of the bacterium was performed using the molecular markers 16S RNA and rpoB, by PCR and sequencing technique, and the evolutionary distance was analyzed by Bayesian phylogenetic inference. No differences were observed in the perimeter and area of larvae treated with EC-35 or Coumaphos. The thickness of the integument decreased a 65% with the EC-35 treatment (6.01 ± 0.6 µm) and of 30% in coumaphos (12.04 ± 1.2 µm) in larvae compared with the control group (18.41 ± 2 µm), while no difference was found in adult ticks. The capillary feeding test and coelom inoculation with EC-35 showed an inhibition of reproductive potential of 99.8 ± 7 and an oviposition Inhibition 97 ± 3.02%. The EC-35 strain was genetically related to Serratia marcescens, concluding that these bacteria caused high mortality, oviposition Inhibition, and integument thinning and drastic loss of histoarchitecture in R. microplus tick larvae.

Zinc Oxide Particles Can Cause Ovarian Toxicity by Oxidative Stress in Female Mice Model.

Introduction: Zinc oxide nanoparticles (ZnO NPs) participate in all aspects of our lives, but with their wide application, more and more disadvantages are exposed. The goal of this study was to investigate the toxicity of ZnO NPs in female mice ovaries and explore its potential mechanism. Methods: In this study, adult female mice were orally exposed to 0, 100, 200, and 400 mg/kg ZnO NPs for 7 days. We explored the underlying mechanisms via the intraperitoneal injection of N-acetyl-cysteine (NAC), an inhibitor of oxidative stress, and salubrinal (Sal), an inhibitor of endoplasmic reticulum (ER) stress. Results: The results indicated that serum estradiol and progesterone levels declined greatly with increasing ZnO NPs dosage. Hematoxylin and eosin (HE) staining revealed increased atretic follicles and exfoliated follicular granulosa cells. Moreover, at the transcriptional level, antioxidant-related genes such as Keap1 and Nrf2, and ER stress-related genes PERK, eIF2α, and ATF4 were markedly upregulated. In addition, the expression of Caspase12, Caspase9, and Caspase3, which are genes related to apoptosis, was also upregulated in all ZnO NPs treatment groups. Serum malondialdehyde (MDA) content was remarkably up-regulated, whereas superoxide dismutase (SOD) activity was down-regulated. The 400 mg/kg ZnO NPs treatment group suffered the most substantial harm. However, ovarian damage was repaired when NAC and Sal were added to this group. Conclusion: ZnO NPs had toxic effects on the ovary of female mice, which were due to oxidative stress, ER stress, and the eventual activation of apoptosis.

Relaxin-3 Ameliorates Diabetic Cardiomyopathy by Inhibiting Endoplasmic Reticulum Stress.

Background: This study is aimed at investigating whether relaxin-3 exhibits protective effects against cardiomyopathy in diabetic rats by suppressing ERS. Methods: Eighty male SD rats were randomly divided into two groups: controls (n = 20) and diabetes (n = 60). The streptozotocin-treated rats were randomly divided into three groups: diabetic group (DM), low-dose relaxin-3 group (0.2 µg/kg/d), and high-dose relaxin-3 group (2 µg/kg/d). The myocardial tissues and collagen fiber were observed by hematoxylin and eosin (H&E) and Masson staining. Serum brain natriuretic peptide (BNP), troponin (TNI), myoglobin, interleukin (IL-17), interleukin (IL)-1α, and tumor necrosis factor (TNF)-α were determined by ELISA. The protein expression of glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the heart tissue of each group was detected by Western blot analysis. Results: (1) HE and Masson staining indicated that relaxin-3 could attenuate myocardial lesions and myocardial collagen volume fraction. (2) BNP, TnI, and myoglobin in the DM group at four and eight weeks were significantly higher than in the controls (P < 0.01). The relaxin-3-treated groups showed significantly reduced serum BNP, TnI, and myoglobin levels compared with the DM group (P < 0.05). (3) IL-17, IL-1α, and TNF-α levels in the DM rats at 4 weeks were higher than in the controls (P < 0.05). Low or high dose of relaxin-3-treated groups showed reduced serum IL-17 and TNF-α levels compared with the DM group at four and eight weeks (P < 0.05). (4) CHOP and GRP78 protein expression was increased in the DM group at four and eight weeks compared with the controls (P < 0.01), and small and large doses of relaxin-3 significantly reduced GRP78 and CHOP protein expression. Conclusions: Exogenous relaxin-3 ameliorates diabetic cardiomyopathy by inhibiting ERS in diabetic rats.

MiR-9a-5p alleviates ventilator-induced lung injury in rats by inhibiting the activation of the MAPK signaling pathway via CXCR4 expression downregulation.

BACKGROUND: Globally, Mechanical ventilation is the most commonly used short-term life support technology. Ventilator-induced lung injury (VILI) is an inflammatory injury caused by mechanical ventilation. MicroRNAs (miRNAs) are considered as new gene regulators that play an important role in lung injury and inflammation. However, the role and mechanism of action of miR-9a-5p in VILI remain unclear. METHODS: Herein, a rat model of VILI was established. To determine the expression levels of miR-9a-5p and CXCR4 mRNA, real-time quantitative polymerase chain reactions (qRT-PCR) were conducted. As well as western blot (WB) and immunofluorescence analyses, we determined the expression of CXCR4, SDF-1 and MAPK signaling pathway-related kinases. Hematoxylin and eosin (H&E) staining and the wet-dry ratio of the lung tissue were used to evaluate organ injury. An enzyme-linked immunosorbent assay (Elisa) and myeloperoxidase (MPO) activity measurements were performed to evaluate the inflammatory response. In addition, double luciferase reporter assays were used to verify the association between miR-9a-5p and CXCR4. RESULTS: The expression of miR-9a-5p was low, whereas that of CXCR4 was high in the lung tissues of VILI rats. The overexpression of miR-9a-5p alleviated the degree of pathological injury in the lung tissues of rats with VILI, downregulating inflammatory cytokine expression and MPO activity. In the VILI rat model, miR-9a-5p targeted the negative regulation of CXCR4, and CXCR4 overexpression to reverse the lung-protective and anti-inflammatory effects of miR-9a-5p overexpression in VILI rats. miR-9a-5p also inhibited the phosphorylation of extracellular signal receptor-activated kinase (ERK), a protein related to the MAPK signaling pathway, by downregulating CXCR4 expression. CONCLUSION: miR-9a-5p can hinder the activation of the MAPK/ERK signaling pathway and reduce inflammatory reactions and lung injury in VILI rats through the targeted regulation of CXCR4 expression. Therefore, miR-9a-5p could serve as an intervention target to supply a new strategy for the care of VILI.

Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response in Fusarium solani-Induced Keratitis via the NF-κB Signaling Pathway.

Purpose: The purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK). Methods: We established corneal stromal cell models and FK mouse models by incubation with Fusarium solani. The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT-PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and TNF-ɑ. Results: Our data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani-induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK. Conclusions: Our research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.

Triptolide improves chondrocyte proliferation and secretion via down-regulation of miR-221 in synovial cell exosomes.

BACKGROUND: Rheumatoid arthritis (RA), the most common type of inflammatory arthritis, can cause bone damage and disability. Triptolide, a prominent treatment for RA, has satisfactory anti-inflammatory effects. However, the mechanism of action of triptolide in RA remains unknown. PURPOSE: This study aimed to explore the molecular mechanisms underlying triptolide-mediated improvements in RA and identify the miRNA pathway responsible for these effects. METHODS: We identified various dysregulated miRNAs associated with RA by mining previously described microarray data and verified and screened these candidates using RT-qPCR. Hematoxylin-eosin staining was then applied to identify pathological changes in the affected joints, and cell counting kit-8 analysis and flow cytometry were employed to examine cell proliferation and apoptosis, respectively. Extracted exosomes were verified using transmission electron microscopy. RESULTS: Our results revealed that the legs of rats with collagen-induced arthritis presented with obvious swelling and bone damage, a high degree of inflammatory cell infiltration into the synovium, and structural changes to the cartilage. Data mining identified 39 dysregulated miRNAs in these tissues, and RT-qPCR further refined these observations to highlight miR-221 as a potential RA biomarker. Subsequent evaluations revealed that fibroblast-like synovial (FLS) cells secrete Exs carrying dysregulated miR-221 in vitro. These Exs mediate miR-221 levels, inflammation, and TLR4/MyD88 signaling via their fusion with chondrocytes, leading to changes in chondrocyte growth and metabolic factor levels. Additionally, the addition of triptolide impaired miR-221 expression, cell proliferation, inflammatory factors, and the protein levels of TLR4/MyD88 in RA-FLS and promoted the apoptosis of FLS. The therapeutic effect of triptolide on miR-221 Exs was reversed by miR-221 inhibitor in both normal and RA FLS. CONCLUSION: Our research shows that effective treatment with triptolide is mediated by its regulation of growth and secretory functions of chondrocytes via the inhibition of miR-221 secretion by FLS, providing a new target and natural medicinal candidate for future RA treatments.

Magnesium yields opposite effects on the nuclear and cytosolic cascades of apoptosis in different rat brain regions.

OBJECTIVE: Magnesium is considered as potential neuroprotective and therapeutic agent, but certain studies have provided evidence of its apoptotic effectiveness in neurons. We aimed to evaluate the possible apoptotic effects of long-term magnesium use in healthy adult rat brains. MATERIALS AND METHODS: Magnesium citrate and magnesium glycinate compounds were administered orally to rats for 8 weeks (36 mg/kg). Expression levels of Bcl-2, Bax and Cyt-C genes were analyzed by real-time polymerase chain reactions (RT-PCR) in the prefrontal cortex, hippocampus and striatum regions. Bcl-2, Bax and CytC protein levels were measured using ELISA kits. Tissue sections were evaluated histopathologically with hematoxylin-eosin staining. RESULTS: Compared to the control group, the magnesium-administered groups indicated gene expression reductions in almost all brain regions; pro-apoptotic Bax, anti-apoptotic Bcl-2 and Cyt-C gene expression levels were reduced. With magnesium, the Bcl-2 and Bax protein levels were increased. Bax/Bcl-2 gene and protein ratio were also increased in the striatum and hippocampus, whereas Cyt-C protein levels were decreased or did not change in the magnesium treated groups. There was no pathological finding in histological evaluation. CONCLUSIONS: Long-term magnesium usage can promote apoptotic cascade in brain tissue by increasing Bax/Bcl-2 ratio. Cyt-C, a prominent factor processing caspase pathway, was decreased or unchanged. In addition, taking into account the histological evaluation, we supposed that the absence of Cyt-C in the cytosol can prevent the subsequent apoptotic pathway. Consequently, we obtained the findings of apoptotic initiation with magnesium in brain, but this cascade seems to be arrested at later stages.