Intraperitoneal Injection Of Normal Saline Research Articles

Oxidative stress causes liver injury via the damage of redox system and the apoptosis induced by mitochondria of broiler

The aim of this study was conducted to explore the effects of oxidative stress on the redox system, mitochondrial function, and apoptosis in liver of broilers. 144 one-day-old male Ross-308 broilers from the same batch had been prepared in advance and were divided into 3 treatments: normal control group (without intraperitoneal injection), normal saline group (intraperitoneal injection of normal saline), and hydrogen peroxide (H2O2) group (intraperitoneal injection of H2O2) with 6 replicates of 8 chickens each. The experimental period was 42 days. The injection volumes were 1.0 mL/kg of Ross-308 broiler body weight. On the 42nd day of the experimental period, two Ross-308 broilers were randomly selected from each cage, a total of 36 broilers were stunned by electric shock and slaughtered to collect liver samples. The results showed that compared with the non-injection group and the injection of saline group, H2O2 exposure elevated the relative weight of liver and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum of Ross-308 broilers (p < 0.05). Meanwhile, compared with the non-injection group and the injection of saline group, the H2O2 exposure increased the levels of reactive oxygen species (ROS), MDA, lipid peroxide (LPO) and protein carbonyl, decreased the activities of total superoxide dismutase (T-SOD), catalase (CAT), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) (p < 0.05). Compared with the non-injection group and the injection of saline group, the H2O2 exposure caused microvesicular steatosis and mitochondrial vacuolisation in the liver tissues of broilers. Additionally, after H2O2 exposure, the levels of nuclear factor erythroid 2–related factor 2 (Nrf2) positive cells were reduced and the relative percentage of hepatocyte apoptosis were increased in the liver tissues of broilers (p < 0.05). The conclusion of this study is that the oxidative stress induced by intraperitoneal injection of H2O2 could induce excessive production of ROS in broiler liver, which damage the antioxidant status and mitochondrial function of liver cells, leading to cell apoptosis. HIGHLIGHTS Oxidative stress can cause severe damage to the liver of broilers. Oxidative stress can mainly damage liver antioxidant function Oxidative stress can induce cell apoptosis by damaging mitochondria in the liver of broilers

Notch1/Akt/Foxo1 Pathway Regulated by Kisspeptin Is Involved in Endometrial Decidualization in Patients With Recurrent Spontaneous Abortion

Kisspeptin, a protein encoded by the KISS1 gene, functions as an essential factor in suppressing tumor growth. The intricate orchestration of cellular processes such as proliferation and differentiation is governed by the Notch1/Akt/Foxo1 signaling pathway, which assumes a central role in maintaining cellular homeostasis. In the specific context of this investigation, the focal point lies in a meticulous exploration of the intricate mechanisms underlying the regulatory effect of kisspeptin on the process of endometrial decidualization. This investigation delves into the interplay between kisspeptin and the Notch1/Akt/Foxo1 signaling pathway, aiming to elucidate its significance in the pathophysiology of recurrent spontaneous abortion (RSA). We enrolled a cohort comprising 45 individuals diagnosed with RSA, who were admitted to the outpatient clinic of the Reproductive Center at the Second Affiliated Hospital of Soochow University between June 2020 and December 2020. On the other hand, an additional group of 50 women undergoing elective abortion at the outpatient clinic of the Family Planning Department during the same timeframe was also included. To comprehensively assess the molecular landscape, Western blot and RT-qPCR were performed to analyze the expression levels of kisspeptin (and its gene KISS1), IGFBP1 (an established marker of decidualization), Notch1, Akt, and Foxo1 within the decidua. Human endometrial stromal cells (hESC) were given targeted interventions, including treatment with siRNA to disrupt KISS1 or exposure to kisspeptin10 (the bioactive fragment of kisspeptin), and were subsequently designated as the siKP group or the KP10 group, respectively. A control group comprised hESC was transfected with blank siRNA, and cell proliferation was meticulously evaluated with CCK8 assay. Following in vitro induction for decidualization across the three experimental groups, immunofluorescence assay was performed to identify differences in Notch1 expression and decidualization morphology between the siKP and the KP10 groups. Furthermore, RT-qPCR and Western blot were performed to gauge the expression levels of IGFBP1, Notch1, Akt, and Foxo1 across the three cell groups. Subsequently, decidualization was induced in hESC by adding inhibitors targeting Notch1, Akt, and Foxo1. The expression profiles of the aforementioned proteins and genes in the four groups were then examined, with hESC induced for decidualization without adding inhibitors serving as the normal control group. To establish murine models of normal pregnancy (NP) and RSA, CBA/J×BALB/c and CBA/J×DBA/2 mice were used. The mice were respectively labeled as the NP model and RSA model. The experimental groups received intraperitoneal injections of kisspeptin10 and kisspeptin234 (acting as a blocker) and were designated as RSA-KP10 and NP-KP234 groups. On the other hand, the control groups received intraperitoneal injections of normal saline (NS) and were referred to as RSA-NS and NP-NS groups. Each group comprised 6 mice, and uterine tissues from embryos at 9.5 days of gestation were meticulously collected for observation of embryo absorption and examination of the expression of the aforementioned proteins and genes. The analysis revealed that the expression levels of kisspeptin, IGFBP1, Notch1, Akt, and Foxo1 were significantly lower in patients diagnosed with RSA compared to those in women with NP (P<0.01 for kisspeptin and P<0.05 for IGFBP1, Notch1, Akt, and Foxo1). After the introduction of kisspeptin10 to hESC, there was an observed enhancement in decidualization capability. Subsequently, the expression levels of Notch1, Akt, and Foxo1 showed an increase, but they decreased after interference with KISS1. Through immunofluorescence analysis, it was observed that proliferative hESC displayed a slender morphology, but they transitioned to a rounder and larger morphology post-decidualization. Concurrently, the expression of Notch1 increased, suggesting enhanced decidualization upon the administration of kisspeptin10, but the expression decreased after interference with KISS1. Further experimentation involved treating hESC with inhibitors specific to Notch1, Akt, and Foxo1 separately, revealing a regulatory sequence of Notch1/Akt/Foxo1 (P<0.05). In comparison to the NS group, NP mice administered with kisspeptin234 exhibited increased fetal absorption rates (P<0.001) and decreased expression of IGFBP1, Notch1, Akt, and Foxo1 (P<0.05). Conversely, RSA mice administered with kisspeptin10 demonstrated decreased fetal absorption rates (P<0.001) and increased expression levels of the aforementioned molecules (P<0.05). It is suggested that kisspeptin might exert its regulatory influence on the process of decidualization through the modulation of the Notch1/Akt/Foxo1 signaling cascade. A down-regulation of the expression levels of kisspeptin could result in suboptimal decidualization, which in turn might contribute to the development or progression of RSA.

Pain experience reduces social avoidance to others in pain: a c-Fos-based functional connectivity network study in mice.

Pain experience increases individuals' perception and contagion of others' pain, but whether pain experience affects individuals' affiliative or antagonistic responses to others' pain is largely unknown. Additionally, the neural mechanisms underlying how pain experience modulates individuals' responses to others' pain remain unclear. In this study, we explored the effects of pain experience on individuals' responses to others' pain and the underlying neural mechanisms. By comparing locomotion, social, exploration, stereotyped, and anxiety-like behaviors of mice without any pain experience (naïve observers) and mice with a similar pain experience (experienced observers) when they observed the pain-free demonstrator with intraperitoneal injection of normal saline and the painful demonstrator with intraperitoneal injection of acetic acid, we found that pain experience of the observers led to decreased social avoidance to the painful demonstrator. Through whole-brain c-Fos quantification, we discovered that pain experience altered neuronal activity and enhanced functional connectivity in the mouse brain. The analysis of complex network and graph theory exhibited that functional connectivity networks and activated hub regions were altered by pain experience. Together, these findings reveal that neuronal activity and functional connectivity networks are involved in the modulation of individuals' responses to others' pain by pain experience.

The role of Cannabinoid receptors in visceral pain sensation of rat: an interventional study

Visceral pain, originating from internal organs, represents a challenging aspect of pain management due to its intricate mechanisms and often debilitating nature. Understanding the underlying pathways involved in visceral pain perception is crucial for developing effective therapeutic strategies. The current study aimed to delve into recent advancements in the understanding of cannabinergic modulation of visceral pain perception, focusing on findings from interventional studies utilizing animal models, particularly rats. A total of 30 male rats aged 3 months, with an average weight of 220 g were randomly divided into 3 groups. The groups contained the control group which received intraperitoneal injection of normal saline, the second group received an intraperitoneal injection of anandamide (2 mg/kg), and the third group received an intraperitoneal injection of tramadol (20 mg/kg). The pain in all groups assessed by acetic acid test. The data obtained from the intraperitoneal injection of anandamide to the rats of the experimental group showed a significant decrease in the amount of perceived visceral pain compared to control group. In addition, the results showed that tramadol injection significantly decreased visceral pain in experimental group 2 compared to the control group. In conclusion, the current study provides an evidence for the involvement of cannabinoid receptors in the modulation of visceral pain sensation in rats.

Downregulation of lncRNA CDKN2B-AS1 attenuates inflammatory response in mice with allergic rhinitis by regulating miR-98-5p/SOCS1 axis

Long non-coding RNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in various diseases has been verified. However, the underlying mechanism of CDKN2B-AS1 contributes to the development of allergic rhinitis (AR) remains unknown. To evaluate the impact of CDKN2B-AS1 on AR, BALB/c mice were sensitized by intraperitoneal injection of normal saline containing ovalbumin (OVA) and calmogastrin to establish an AR model. Nasal rubbing and sneezing were documented after the final OVA treatment. The concentrations of IgE, IgG1, and inflammatory elements were quantified using ELISA. Hematoxylin and eosin (H&E) staining and immunofluorescence were used to assess histopathological variations and tryptase expression, respectively. StarBase, TargetScan and luciferase reporter assays were applied to predict and confirm the interactions among CDKN2B-AS1, miR-98-5p, and SOCS1. CDKN2B-AS1, miR-98-5p, and SOCS1 levels were assessed by quantitative real-time PCR (qRT-PCR) or western blotting. Our results revealed that CDKN2B-AS1 was obviously over-expressed in the nasal mucosa of AR patients and AR mice. Down-regulation of CDKN2B-AS1 significantly decreased nasal rubbing and sneezing frequencies, IgE and IgG1 concentrations, and cytokine levels. Furthermore, down-regulation of CDKN2B-AS1 also relieved the pathological changes in the nasal mucosa, and the infiltration of eosinophils and mast cells. Importantly, these results were reversed by the miR-98-5p inhibitor, whereas miR-98-5p directly targeted CDKN2B-AS1, and miR-98-5p negatively regulated SOCS1 level. Our findings demonstrate that down-regulation of CDKN2B-AS1 improves allergic inflammation and symptoms in a murine model of AR through the miR-98-5p/SOCS1 axis, which provides new insights into the latent functions of CDKN2B-AS1 in AR treatment.

MPTP- Induced BALB/C Mice Recapitulate Compensatory Parkinson’s-Like Motor Features

This study aimed to assess motor responses and associated pathological changes caused by MPTP neurotoxicity in Balb/c mice. Male mice (13 weeks old, 25-30 g) were divided into four groups and received intraperitoneal injections of normal saline or different doses of MPTP-HCl for five consecutive days. Body weight was monitored, and behavioral tests were conducted. Histological examination with H&amp;E staining was performed on the striatum and substantia nigra. Contrary to expectations, MPTP-treated mice showed increased locomotor activity in the open field test, covering a greater distance and exhibiting more rearing compared to control mice (p&lt;0.05). The catalepsy test also showed lower catalepsy scores in the MPTP-treated group (p&lt;0.05). However, the pole test did not indicate the presence of MPTP-induced bradykinesia (p&gt;0.05). Similarly, the traction and hang tests showed no significant effects of MPTP on motor balance or muscle strength (p&gt;0.05). Among the MPTP-treated groups, the 30 mg/kg MPTP-HCl group displayed the most severe pathological changes, including reactive gliosis, as observed in histological examination. In conclusion, the subacute MPTP mouse model used in this study did not exhibit noticeable motor deficits or significant weight loss in Balb/c mice, possibly due to subthreshold dopamine depletion compensatory mechanisms. This model could provide valuable insights into the compensatory mechanisms involved in Parkinson's disease.

Effect of nerve growth factor on elderly degenerative knee osteoarthritis pain

To explore effect of nerve growth factor (NGF) antibody on knee osteoarthritis (KOA) pain model was evaluated by in vitro model. Thirty male SPF rats aged 28-week-old were divided into blank group (10 rats with anesthesia only). The other 20 rats were with monoiodoacetate (MIA) on the right knee joint to establish pain model of OA, and were randomly divided into control group (injected intraperitoneal injection of normal saline) and treatment group (injected anti-NGF) intraperitoneal after successful modeling, and 10 rats in each group. All rats were received retrograde injection of fluorogold (FG) into the right knee joint. Gait was assessed using catwalk gait analysis system before treatment, 1 and 2 weeks after treatment. Three weeks after treatment, right dorsal root ganglia (DRG) were excised on L4-L6 level, immunostained for calcitonin gene-related peptide (CGRP), and the number of DRGS was counted. In terms of gait analysis using cat track system, duty cycle, swing speed and print area ratio in control and treatment group were significantly reduced compared with blank group (P<0.05). Compared with control group, duty cycle and swing speed of treatment group were significantly improved (P<0.05), and there was no significant difference in print area ratio between treatment group and blank group (P>0.05). The number of FG-labeled DRG neurons in control group was significantly higher than that in treatment group and blank group (P<0.05). The expression of CGRP in control group was up-regulated, and differences were statistically significant compared with treatment group (P<0.05). Intraperitoneal injection of anti-NGF antibody inhibited gait injury and upregulation of CGRP in DRG neurons. The results suggest that anti-nerve growth factor therapy may be of value in treating knee pain. NGF may be an important target for the treatment of knee OA pain.

Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn

To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

Angong Niuhuang Wan ameliorates LPS-induced cerebrovascular edema by inhibiting blood‒brain barrier leakage and promoting the membrane expression of AQP4.

Angong Niuhuang Wan (AGNHW), developed during the Qing dynasty (18th century) for the treatment of consciousness disturbances caused by severe infections, has been used to treat brain edema caused by ischemia‒reperfusion. However, it remains unclear whether AGNHW can ameliorate vascular-origin brain edema caused by lipopolysaccharides (LPS). This study explored the ameliorative effects of AGNHW on LPS-induced cerebrovascular edema in mice, as well as the potential underlying mechanisms. A cerebrovascular edema model was established in male C57BL/6N mice by two intraperitoneal injections of LPS (15mg/kg), at 0 and 24h. AGNHW was administered by gavage at doses of 0.2275g/kg, 0.455g/kg, and 0.91g/kg, 2h after LPS administration. In control mice, normal saline (NS) or AGNHW (0.455g/kg) was administered by gavage 2h after intraperitoneal injection of NS. The survival rate, cerebral water content, cerebral venous FITC-dextran leakage, Evans blue extravasation, and expression of vascular endothelial cadherin (VE-cadherin), zonula occludens-1 (ZO-1), claudin-5, phosphorylated caveolin-1 (CAV-1), and cytomembrane and cytoplasmic aquaporin 1 (AQP1) and aquaporin 4 (AQP4) were evaluated. The cerebral tissue phosphoproteome, blood levels of AGNHW metabolites, and the relationships between these blood metabolites and differentially phosphorylated proteins were analyzed. AGNHW inhibited the LPS-induced decrease in survival rate, increase in cerebral water content, decrease in VE-Cadherin expression and increase in phosphorylated CAV-1 (P-CAV-1). AGNHW treatment increased the expression of AQP4 on astrocyte membrane after LPS injection. AGNHW also inhibited the LPS-induced increases in the phosphorylation of 21 proteins, including protein kinase C-α (PKC-α) and mitogen-activated protein kinase 1 (MAPK1), in the cerebral tissue. Eleven AGNHW metabolites were detected in the blood. These metabolites might exert therapeutic effects by regulating PKC-α and MAPK1. AGNHW can ameliorate cerebrovascular edema caused by LPS. This effect is associated with the inhibition of VE-Cadherin reduction and CAV-1 phosphorylation, as well as the upregulation of AQP4 expression on the astrocyte membrane, following LPS injection.

Preliminary Investigation of the Molecular Mechanism of Empagliflozin Suppressing Gastric Cancer Through Mammalian Target of Rapamycin

To predict the intervention targets of empagliflozin (EMPA), a specific inhibitor of sodium-glucose cotransporter 2 (SGLT2), in gastric adenocarcinoma through comprehensive network pharmacology, and to validate the effects and the molecular mechanisms of EMPA through cellular and molecular biology experiments. Bioinformatics analysis of gastric adenocarcinoma was conducted to assess the correlation between gastric adenocarcinoma prognosis and SGLT2 expression. Network pharmacology was utilized to identify shared targets of EMPA and gastric adenocarcinoma. AGS cells, a human gastric adenocarcinoma cells line, were incubated with EMPA at different concentrations for 24 h and, then, cell proliferation was assessed using the CCK8 assay. After AGS cells were incubated with EMPA at the doses of 0, 3, and 6 mmol/L, real-time cell analysis (RTCA) and 5-ethynyl-2-deoxyuridine (EdU) incorporation were used to evaluate EMPA's inhibitory effects on the proliferation of the AGS cells. In addition, wound healing and Transwell assays were performed to assess the inhibitory effect of EMPA on the migration and invasion of the APC cells and Western blot analysis was conducted to examine the expression of mammalian target of rapamycin (mTOR) and phosphorylated mTOR (p-mTOR). BALB/c (nu/nu) nude mice were implanted with 5×106 AGS cells in the axilla. The mice were divided into three groups, a control group, a low-dose group, and a high-dose group, each consisting of 7 mice. After one week, the control group received daily intraperitoneal injections of normal saline, while the low-dose group and high-dose group received daily intraperitoneal injections of EMPA at the doses of 3 mg/kg and 5 mg/kg, respectively. The tumor volume was measured one week after the drug intervention started. Gastric adenocarcinoma patients with low expression of SGLT2 exhibited longer survival time and higher survival rate than those with high expression of SGLT2 did. A total of 104 EMPA-related potential targets and 2028 targets associated with gastric adenocarcinoma were identified. Among these, 45 targets associated with gastric adenocarcinoma overlapped with potential targets of EMPA. Further analysis revealed 10 relevant pathways and 4 core genes. The core genes were cyclin-dependent kinase 4 (CDK4), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), mTOR, and cyclin E1 (CCNE1). CCK-8 assay revealed that EMPA at concentrations ranging from 0.39 to 50 mmol/L effectively inhibited the proliferation of AGS cells. RTCA results indicated a downward shift in the cell growth curve. In comparison to the findings for the control group, EdU assay demonstrated that EMPA at the concentrations of 3 mmol/L and 6 mmol/L significantly inhibited AGS cell proliferation (P<0.05). Results from wound healing and Transwell assays indicated a decrease in the levels of cell migration and invasion (P<0.05) and, notably, there was a significant difference between the high and low-dose EMPA groups (P<0.05). Western blot showed no statistically significant difference in the expression of total mTOR protein between the groups. However, the expression of p-mTOR in the 3 mmol/L and 6 mmol/L EMPA groups decreased compared to that of the control group (P<0.05), with the 6 mmol/L EMPA group exhibiting a more pronounced reduction (P<0.05). Nude mice xenograft tumor experiment demonstrated that, compared to that of the control group, the tumor volumes in the EMPA-treatment groups were significantly reduced (P<0.05), with the high-dose group showing a more pronounced reduction (P<0.05). EMPA inhibits the abnormal proliferation and migration of gastric adenocarcinoma cells, potentially through the modulation of mTOR protein activation. This study provides new potential medication and intervention targets for gastric adenocarcinoma treatment.

Trimethylamine N-Oxide Induces Renal Fibrosis Through the PI3K/AKT/SREBP1 Pathway

To investigate the role and mechanism of trimethylamine N-oxide (TMAO), a uremic toxin, in renal fibrosis. A total of 20 male BALB/c mice were randomly and evenly assigned to a Control group and a TMAO group. Mice in the Control group received intraperitoneal injection of normal saline, while mice in the TMAO group received intraperitoneal injection of TMAO (20 mg/[kg·d]). The injection was given once a day for 8 weeks. Histopathology and fibrosis of kidney were observed by H&E staining and Masson staining. Immunohistochemistry was performed to determine the levels of alpha smooth muscle actin (α-SMA), recombinant human fibronectin fragment (Fibronectin), and sterol-regulatory element binding protein 1 (SREBP1). Western blot was performed to determine α-SMA, SREBP1, phosphatidylinositol 3 kinase (PI3K), phospho-phosphatidylinositol 3 kinase (p-PI3K), protein kinase B (PKB, also known as AKT), and phospho-AKT (p-AKT) protein levels. HK2 cells were treated with SREBP1 small interfering RNA (siRNA) and PI3K/AKT inhibitor, respectively, and the reversal of the effects of TMAO was examined. Animal experiments showed that, compared with the Control group, the mice treated with TMAO experienced pathological damage and fibrosis of the kidney tissue and the expression levels of fibrosis markers, α-SMA and Fibronectin, in the kidney were increased (all P<0.05). According to the findings from further investigation, the TMAO-treatment group showed increased expression of SREBP1 and an up-regulation of PI3K phosphorylation ratio and AKT phosphorylation ratio compared with those of the Control group (all P<0.05). Cell experiments produced results similar to those of the animal experiment. After siRNA interference with SREBP1 expression, the expression levels of fibrosis marker proteins decreased (P<0.05). Besides, the high expression of SREBP1 caused by TMAO was inhibited after HK2 cells were incubated with LY294002, a PI3K-AKT pathway inhibitor (P<0.05). TMAO may induce renal fibrosis by promoting the PI3K/AKT/SREBP1 pathway.

Electroacupuncture combined with bone marrow mesenchymal stem cell transplantation promotes repair of thin endometrium by regulating SDF-1/CXCR4 signaling

It is to explore, based on stromal cell derived factor 1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) signal axis, whether the electroacupuncture (EA) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation can promote thin endometrium regeneration and improve endometrial receptivity, so as to further study its mechanisms underlying improvement of promoting BMSCs homing to repair thin endometrium. Thirty matured female SD rats were randomly divided into normal control , model , BMSCs transplantation (BMSCs), BMSCs+AMD3100 (a specific antagonist of CXCR4, BMSCs+AMD3100), BMSCs+EA, and BMSCs+EA+AMD3100 groups, with 5 rats in each group. The thin endometrial model was established by intrauterine injection of 95% ethanol during the period of estrus. Rats of the model group received intravenous injection of PBS solution (tail vein) on day 1, 3 and 7 of modeling and intraperitoneal injection of normal saline once daily for 3 estrous cycles. Rats of the BMSCs group received intravenous injection of BMSCs suspension on day 1,3 and 7 of modeling, and those of the BMSCs+EA group received BMSCs transplantation and EA stimulation. EA (2 Hz/15 Hz, 1 mA) was applied to "Guanyuan" (CV4) and bilateral "Sanyinjiao"(SP9), "Zigong" (EX-CA1) for 15 min, once daily for 3 estrous cycles. Rats of the BMSCs+AMD3100 group received intravenous injection of BMSCs suspension (1×106/mL) and intraperitoneal injection of AMD3100 (5 mg/kg), and those of the BMSCs+EA+AMD3100 group received administration of BMSCs, AMD3100 and EA, with both groups being once daily for 3 estrous cycles. H.E. staining was used to observe histopathological changes of endometrium tissues, and immunohistochemistry was used to detect the expressions of cytokeratin (CK19) and vimentin in endometrium (for evaluating the damage and repair of endometrium). The expression levels of homeobox A10 (HOXA10), leukemia inhibitory factor (LIF), SDF-1 and CXCR4 proteins were detected by Western blot, and those of SDF-1 and CXCR4 mRNAs in the endometrium detected by real-time PCR. In comparison with the normal control group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression leve-ls of HOXA10, LIF and CXCR4 proteins and CXCR4 mRNA were significantly down-regulated (P<0.01), and the expression levels of SDF-1 protein and mRNA significantly up-regulated (P<0.05) in the model group. Compared with the model group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, and the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs group, and the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA, and SDF-1 protein and mRNA in the BMSCs+EA group were significantly up-regulated (P<0.05, P<0.01). Compared to the BMSCs group, the number of endometrial glands, and the expression levels of LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA group were up-regulated (P<0.01, P<0.05)； the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+AMD3100 group were down-regulated (P<0.01). Compared to the BMSCs+EA group, the number of endometrial glands, the immunoactivity of CK19 and vimentin, the expression levels of HOXA10, LIF, CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA+AMD3100 group were down-regulated (P<0.01). Results of H.E. staining showed thin endometrium with absence of epithelial cells, and sparse glands and blood vessels, with smaller glandular cavity in the model group, which was relative milder in BMSCs and BMSCs+EA groups. EA can promote the transfer of transplanted BMSCs to the damaged site through SDF-1/CXCR4 signaling related stem cell homing, thereby promoting thin endometrial regeneration, repairing endometrial injury, and improving endometrial tolerance in rats with thin endometrium.

Melatonin Ameliorates Cyclophosphamide-Induced Spermatogenesis Disorder by Reducing Pyroptosis

As a chemotherapeutic drug, cyclophosphamide (CP) has a negative impact on male fertility due to its reproductive toxicity. Melatonin (Mel) promotes the male reproductive system and increases testosterone synthesis. This study is aimed at exploring the molecular mechanism of Mel as a protector of male fertility against CP-induced cytotoxicity. A CP toxicity model was established in adult ICR male mice by intraperitoneal injection of 100 mg/kg CP every other day for a week. Protective effects of Mel on the testis from CP-induced damage were evaluated using four groups of ICR male mice that received intraperitoneal injections of normal saline, 100 mg/kg CP, 10 mg/kg Mel, or the same dosage of CP and Mel, respectively. Testis morphology was observed by hematoxylin and eosin (HE) staining. Sperm quality parameters were evaluated, and sperm function was studied by in vitro fertilization (IVF). Proliferation, meiosis, and pyroptosis markers were examined by western blot. Results showed that CP treatment induced testis toxicity in a time-dependent manner with the most severe damage to the testis at two weeks post CP treatment. CP-treated mice showed reduced testicular weight and impaired spermatogenesis by downregulating PCNA and SYCP3, reduced serum testosterone levels, decreased sperm counts and motility, increased seminiferous tubule vacuolization, and oxidative damage to spermatogenic cells. All these effects, apart from testicular weight, could be ameliorated by Mel administration. The IVF experiment revealed that CP treatment reduced the rates of sperm fertilization and blastocyst development, which were also enhanced by Mel. Mel-treated mice also showed increased expression of proliferation-associated protein PCNA and meiosis-associated proteins REC8, STRA8, and SYCP3, which were all reduced by CP. Furthermore, Mel inhibited the pyroptosis of spermatogenic cells by reducing GSDMD and IL18 expression. In conclusion, this study indicated that Mel might protect the testis from CP-induced DNA damage to germ cells through the alleviation of pyroptosis.

Tissue Inhibitor of Metalloproteinase 3 as a potential staging biomarker in hepatocellular carcinoma

Objective: To assess the potential role of tissue inhibitor of metalloproteinase 3 as a staging marker of hepatocellular carcinoma.Method: The experimental study was conducted at Faculty of Pharmacy, Kafrelsheikh University, Egypt, from December 2020 to March 2022 after approval from the Faculty of Pharmacy, Kafrelsheikh University, Egypt, and comprised male albino rats. The subjects were divided into 4 groups. The control group was administrated a single intraperitoneal injection of normal saline, while the other groups were generated post-induction of hepatocellular carcinoma. The induction was done by injecting rats intraperitoneally with a single dose of 200mg/kg diethyl nitrosamine, followed by the administration of 0.05% phenobarbital sodium in drinking water daily. Rats were euthanised at 8, 16 and 24 weeks after the injection to obtain three groups related to the 3 stages of hepatocellular carcinoma. Serum was used for measuring the alpha protein level. Liver homogenates were used for the assessmentof the hepatic tissue inhibitor of metalloproteinase 3 expression, B-cell lymphoma 2-associated X protein expression, and the hepatic content of matrix metalloproteinase -9 and cyclin D1. Data was analysed using Graph Prism 8.Results: Of the 24 rats with weight range 120-130g, 6(25%) were in each of the 4 groups. The relative protein and messenger ribonucleic acid tissue inhibitor of metalloproteinase 3 expressions were significantly decreased in the intervention groups compared to the control group, with more decline as the hepatocellular carcinoma stage increased. The matrix metalloproteinase -9 and cyclin D1 concentrations and the relative hepatic protein Ki67 expression were significantly raised in the intervention groups compared to the control group (p&lt;0.05). The relative expression of hepatic B-cell lymphoma 2-associated X protein was markedly decreased in the intervention groupscompared to the control group (p&lt;0.05).Conclusion: Tissue inhibitor of metalloproteinase 3 might be a promising diagnostic and staging biomarker in hepatocellular carcinoma.Keywords: Carcinoma, Hepatocellular, Neoplasms, Fetoproteins, Metalloproteinase, Cyclin, Antigen, Phenobarbital, Nitrosamines.

Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway

Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3(PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine(8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1(FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.

Lycopene attenuates D-galactose-induced cognitive decline by enhancing mitochondrial function and improving insulin signaling in the brains of female CD-1 mice

The incidence of neurodegenerative diseases is severely increasing with ageing. Lycopene (LYC), a carotenoid pigment, has been reported to have antioxidant, anti-inflammatory and neuroprotective properties. In the present study, we aimed to investigate the ameliorative effect of LYC on D-galactose (D-gal) induced cognitive defects and the underlying mechanisms. Forty-five female CD-1 mice (2 months old) were separated into three groups to be fed with either a normal diet or a LYC diet (0.03%, w/w, mixed into normal diet). Meanwhile, the mice were treated by intraperitoneal injection of normal saline or D-gal 150 mg/kg/day for 8 weeks. The behavioral test results indicated that LYC alleviated D-gal induced cognitive impairments. LYC ameliorated brain ageing by decreasing the number of SA-β-gal- stained neurons, downregulating the protein expression of the cellular senescence associated genes P19/P21/P53, increasing the activities of the antioxidant enzymes GSH and SOD, downregulating the level of ROS, inhibiting the activation of MAPKs signaling and downregulating the levels of the inflammatory cytokines IL-1β and TNFɑ in mouse brains. LYC ameliorated synaptic dysfunction by increasing the expression of the neurotrophic factor BDNF and synaptic proteins. Moreover, LYC attenuated D-gal-induced mitochondrial morphological damage, and promoted the expression of mitochondrial functional proteins. LYC also promoted insulin signal transduction in mouse brains through the regulation of IRS-1/AKT/GSK3β signaling.

Tissue Inhibitor Of Metalloproteinase 3 As A Potential Staging Biomarker In Hepatocellular Carcinoma.

To assessthe potential role of tissue inhibitor of metalloproteinase 3 as a staging marker of hepatocellular carcinoma. The experimental study was conducted at Faculty of Pharmacy, Kafrelsheikh University, Egypt, from December 2020 to March 2022 after approval from the Faculty of Pharmacy, Kafrelsheikh University, Egypt, and comprised male albino rats. The subjects were divided into 4 groups. The control group was administrated a single intraperitoneal injection of normal saline, while the other groups were generated post-induction of hepatocellular carcinoma. The induction was done by injecting rats intraperitoneally with a single dose of 200mg/kg diethyl nitrosamine, followed by the administration of 0.05% phenobarbital sodium in drinking water daily. Rats were euthanised at 8, 16 and 24 weeks after the injection to obtain three groups related to the 3 stages of hepatocellular carcinoma. Serum was used for measuring the alpha protein level. Liver homogenates were used for the assessment of the hepatic tissue inhibitor of metalloproteinase 3 expression, B-cell lymphoma 2-associated X protein expression, and the hepatic content of matrix metalloproteinase -9 and cyclin D1. Data was analysed using Graph Prism 8. Of the 24 rats with weight range 120-130g, 6(25%) were in each of the 4 groups. The relative protein and messenger ribonucleic acid tissue inhibitor of metalloproteinase 3 expressions were significantly decreased in the intervention groups compared to the control group, with more decline as the hepatocellular carcinoma stage increased. The matrix metalloproteinase -9 and cyclin D1 concentrations and the relative hepatic protein Ki67 expression were significantly raised in the intervention groups compared to the control group (p<0.05). The relative expression of hepatic B-cell lymphoma 2-associated X protein was markedly decreased in the intervention groups compared to the control group (p<0.05). Tissue inhibitor of metalloproteinase 3 might be a promising diagnostic and staging biomarker in hepatocellular carcinoma.

Potential roles of 4HNE-adducted protein in serum extracellular vesicles as an early indicator of oxidative response against doxorubicin-induced cardiomyopathy in rats

Late-onset cardiomyopathy is becoming more common among cancer survivors, particularly those who received doxorubicin (DOXO) treatment. However, few clinically available cardiac biomarkers can predict an unfavorable cardiac outcome before cell death. Extracellular vesicles (EVs) are emerging as biomarkers for cardiovascular diseases and others. This study aimed to measure dynamic 4-hydroxynonenal (4HNE)-adducted protein levels in rats treated chronically with DOXO and examine their link with oxidative stress, antioxidant gene expression in cardiac tissues, and cardiac function. Twenty-two male Wistar rats were randomly assigned to receive intraperitoneal injection of normal saline (n = 8) or DOXO (3 mg/kg, 6 doses, n = 14). Before and after therapy, serum EVs and N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were determined. Tunable resistive pulse sensing was used to measure EV size and concentration. ELISA was used to assess 4HNE-adducted protein in EVs and cardiac tissues. Differential-display reverse transcription-PCR was used to quantitate cardiac Cat and Gpx1 gene expression. Potential correlations between 4HNE-adducted protein levels in EVs, cardiac oxidative stress, antioxidant gene expression, and cardiac function were determined. DOXO-treated rats showed more serum EV 4HNE-adducted protein than NSS-treated rats at day 9 and later endpoints, whereas NT-proBNP levels were not different between groups. Moreover, on day 9, surviving rats' EVs had higher levels of 4HNE-adducted protein, and these correlated positively with concentrations of heart tissue 4HNE adduction and copy numbers of Cat and Gpx1, while at endpoint correlated negatively with cardiac functions. Therefore, 4HNE-adducted protein in serum EVs could be an early, minimally invasive biomarker of the oxidative response and cardiac function in DOXO-induced cardiomyopathy.

A novel fusion protein consisting of anti-ANGPTL3 antibody and interleukin-22 ameliorates diabetic nephropathy in mice.

The pathogenic mechanisms of diabetic nephropathy (DN) include podocyte injury, inflammatory responses and metabolic disorders. Although the antagonism of Angiopoietin-like protein 3 (ANGPTL3) can alleviate proteinuria symptoms by inhibiting the activation of integrin αvβ3 on the surface of podocytes, it can not impede other pathological processes, such as inflammatory responses and metabolic dysfunction of glucolipid. Interleukin-22 (IL-22) is considered to be a pivotal molecule involved in suppressing inflammatory responses, initiating regenerative repair, and regulating glucolipid metabolism. Genes encoding the mIL22IgG2aFc and two chains of anti-ANGPTL3 antibody and bifunctional protein were synthesized. Then, the DN mice were treated with intraperitoneal injection of normal saline, anti-ANGPTL3 (20 mg/kg), mIL22Fc (12 mg/kg) or anti-ANGPTL3 /IL22 (25.3 mg/kg) and irrigation of positive drug losartan (20mg/kg/d) twice a week for 8 weeks. In this research, a novel bifunctional fusion protein (anti-ANGPTL3/IL22) formed by the fusion of IL-22 with the C-terminus of anti-ANGPTL3 antibody exhibited favorable stability and maintained the biological activity of anti-ANGPTL3 and IL-22, respectively. The fusion protein showed a more pronounced attenuation of proteinuria and improved dysfunction of glucolipid metabolism compared with mIL22Fc or anti-ANGPTL3. Our results also indicated that anti-ANGPTL3/IL22 intervention significantly alleviated renal fibrosis via inhibiting the expression of the inflammatory response-related protein nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) p65 and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome. Moreover, transcriptome analysis revealed the downregulation of signaling pathways associated with injury and dysfunction of the renal parenchymal cell indicating the possible protective mechanisms of anti-ANGPTL3/IL22 in DN. Collectively, anti-ANGPTL3/IL22 bifunctional fusion protein can be a promising novel therapeutic strategy for DN by reducing podocyte injury, ameliorating inflammatory response, and enhancing renal tissue recovery.

Astragalus polysaccharide protects against blood-brain barrier damage in MCAO rats by inhibiting P2X7R channel

To investigate the protective effect of astragalus polysaccharide (APS) against blood-brain barrier in a rat model of middle cerebral artery occlusion (MCAO) and the role of P2X7R channel in the protective mechanism. In rat microglial cell models of oxygen and glucose deprivation (OGD) or ATP treatment, the formation of blood-brain barrier in vitro was assessed using the leak test, and the effect of APS on the permeability of the blood-brain barrier was determined using LC-MS. In 12 SD rats, MCAO model was established followed by treatment with intraperitoneal injection of normal saline (n= 6) or APS (45 mg/kg, n=6) for 3 consecutive days, with another 6 rats without MCAO receiving saline injections as the control group. The permeability of the blood-brain barrier of the rats was evaluated by determining Evans blue (EB) extravasation, and ATP content in the brain tissue was detected using ELISA; the expression levels of matrix metalloproteinase-9 (MMP-9) and P2X7R in the brain tissue were detected with Western blot. In the in vitro cell model of OGD or ATP treatment, APS treatment obviously promoted the repair of blood-brain barrier integrity. In the rat models, the EB content in the brain tissue and the blood-brain barrier permeability increased significantly in MCAO+saline group and MCAO+APS group as compared with those in the control group (P < 0.01). Compared with saline treatment, APS treatment significantly decreased EB content in the brain tissue and improved the blood-brain barrier permeability in the MCAO rats (P < 0.05). MCAO caused a significant reduction of ATP content and obviously increased the expression levels of MMP-9 and P2X7R in the brain tissue of the rats (P < 0.01), and these changes were significantly alleviated after APS treatment (P < 0.01 or 0.05). APS can protect the brain tissue of MCAO rats by stabilizing the internal environment, down-regulating the expression of MMP-9 and improving the permeability of blood-brain barrier under cerebral ischemia and hypoxia, and its mechanism may involve the inhibition of P2X7R channel.