Peritoneal Injection Research Articles

Deletion of Fgfr2 in Ductal Basal Epithelium With Tamoxifen Induces Obstructive Meibomian Gland Dysfunction.

Fibroblast growth factor receptor 2 (Fgfr2) is crucial for the homeostasis of meibomian gland (MG). However, the role of Fgfr2 in MG ductal epithelial progenitors remains to be delineated. Herein, we created a new transgenic mouse model with conditional deletion of Fgfr2 from MG ductal progenitors and investigated the cell-specific role in the pathogenesis of obstructive meibomian gland dysfunction. Peritoneal injection of tamoxifen (TAM) at 50 µg/gm for three consecutive days was performed to induce conditional deletion of Fgfr2 in two-month-old Krt5Fgfr2CKO or Krt5Fgfr2CKO-mTmG mice. Phenotypes of MG after Fgfr2 deletion were monitored by meibography, lipid staining, and immunostaining against keratin-6a in MG whole mounts. Lineage tracing of the Krt5+ progenitors of MG and biomarkers for ductal differentiation and proliferation were also examined by immunostainings. The Krt5Fgfr2CKO mice developed extensive ductal occlusion and acinar atrophy at day 10 after TAM administration. Robust thickening of ductal epithelium with abnormal differentiation and proliferation of ductal basal meibocytes were observed in the MGs of Krt5Fgfr2CKO mice. In Krt5Fgfr2CKO-mTmG mice, the Krt5+ progenitors and its progeny were labeled by EGFP after Fgfr2 depletion by TAM with evident expansion of the suprabasal and superficial layers of MG ductal epithelium when compared with the controls. Our results substantiated the crucial role of Fgfr2 in homeostasis of the MG ductal epithelium. Deletion of Fgfr2 affects the MG ductal basal progenitors by impacting the differentiation of ductal meibocytes and the maintenance of acinar meibocytes, which are likely the underlying pathogenesis of obstructive MGD.

The lipooligosaccharide of the gut symbiont Akkermansia muciniphila exhibits a remarkable structure and TLR signaling capacity

The cell-envelope of Gram-negative bacteria contains endotoxic lipopolysaccharides (LPS) that are recognized by the innate immune system via Toll-Like Receptors (TLRs). The intestinal mucosal symbiont Akkermansia muciniphila is known to confer beneficial effects on the host and has a Gram-negative architecture. Here we show that A. muciniphila LPS lacks the O-polysaccharide repeating unit, with the resulting lipooligosaccharide (LOS) having unprecedented structural and signaling properties. The LOS consists of a complex glycan chain bearing two distinct undeca- and hexadecasaccharide units each containing three 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residues. The lipid A moiety appears as a mixture of differently phosphorylated and acylated species and carries either linear or branched acyl moieties. Peritoneal injection of the LOS in mice increased higher gene expression of liver TLR2 than TLR4 (100-fold) and induced high IL-10 gene expression. A. muciniphila LOS was found to signal both through TLR4 and TLR2, whereas lipid A only induced TLR2 in a human cell line. We propose that the unique structure of the A. muciniphila LOS allows interaction with TLR2, thus generating an anti-inflammatory response as to compensate for the canonical inflammatory signaling associated with LOS and TLR4, rationalizing its beneficial host interaction.

Effects of the Thiol Methyltransferase Inhibitor (±)-2,3-Dichloro-α-Methylbenzylamine (DCMB) on the Pharmacokinetics and Metabolism of Vicagrel in Rats.

P2Y12 receptor inhibitors are commonly used in clinical antiplatelet therapy, typically alongside other medications. Vicagrel, a promising P2Y12 receptor inhibitor, has submitted a new drug marketing application to the United States Food and Drug Administration. Its primary metabolites and some metabolic pathways are identical to those of clopidogrel. The aim of this study was to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine (DCMB) on the metabolism and pharmacokinetics of vicagrel. In vitro incubation with human and rat liver microsomes revealed that DCMB significantly inhibited the methylation of vicagrel's thiol metabolite M15-1. Rats were orally administered 6 mg/kg [14C]vicagrel (100 μCi/kg) 1 hour after peritoneal injection with or without DCMB (80 mg/kg). Compared with the control group, the plasma of DCMB-pretreated rats exhibited maximum plasma concentration (C max) decrease and time to reach C max (T max) delay for all vicagrel-related substances, the methylation product of the thiol metabolite (M9-2), and the derivatization product of the active thiol metabolite (MP-M15-2). However, no significant changes in area under the curve (AUC) or half-life (t 1/2) were observed. DCMB had negligible effect on the total radiological recovery of vicagrel within 72 hours, although the rate of vicagrel excretion slowed down within 48 hours. DCMB had a negligible impact on the metabolic pathway of vicagrel. Overall, the present study found that DCMB did not significantly affect the total exposure, metabolic pathways, metabolite profiles, or total excretion rates of vicagrel-related metabolites in rats, but led to C max decrease, T max delay, and slower excretion rate within 48 hours. SIGNIFICANCE STATEMENT: This study used liquid chromatography-tandem mass spectrometry combined with radiolabeling technology to investigate the effects of the thiol methyltransferase inhibitor (±)-2,3-dichloro-α-methylbenzylamine on the absorption, metabolism, and excretion of vicagrel in rats. This work helps to better understand the in vivo metabolism of active thiol metabolites of P2Y12 inhibitors such as clopidogrel, vicagrel, etc.

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid–liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

PHARMACOLOGY OF SPONDIAS MOMBIN IN LIVER CANCER TREATMENT AND TOXICITY EVALUATION

Cancer is among the major causes of death in the world. Spondias mombin is a natural product being accepted as medication for many diseases. This research aimed at evaluating the toxicity potential and pharmacology properties of Spondias mombin leaves in liver cancer treatment. Spondias mombin leaves were shade dried at room temperature and size reduced to powder and extracted using cold maceration. Phytochemical screening of the plant extract was conducted. Six weeks of healthy mice; weighing 22-25g were used. For acute toxicity studies, ten mice were grouped into a normal control group and another group received 2000mg/kg of the extract. The animals were observed closely for 14 days. Twenty-four mice were divided into 4 groups. One group served as control and received only saline at 10mls/kg and the other groups received one percent diethylnitrosamine (DEN) given via peritoneal injection to the mice weekly at a dose of 35mg/kg for six weeks. The remaining three groups received 250 mg/kg, 500 mg, and doxorubicin 2 mg/kg after four weeks of induction. The treatment commenced with the extract while still giving the DEN for cancer induction. The phytochemical analysis shows the presence of alkaloids, cardiac glycosides, saponins, tannins, phenolic compounds, steroids, flavonoids, and terpenoids, and the absence of carbohydrates anthraquinones. Animals exposed to the extract and observed for 14 days exhibited no obvious sign of toxicity with zero mortality during the period of observation. There is a slight weight change among the control group between 20.8±3.56 to 25.63 2.60.The creatinine, CL, and HCO3 of the control significantly (P˂0.005) differed from the study groups. There was a slight increase in weight after the first dose, though a steady decrease was noticed following the second, third, and fourth doses of DEN in the mice. ALT and AST were higher in the extract-treated groups compared to both Dox-treated and saline-treated groups. The extract was shown to have no obvious physical sign of toxicity and it doesn’t cause mortality in all the tested animals. Ethanol extract of the plant reversed the loss in weight, deterioration in liver enzymes, and protein production to an extent similar to that of standard anti-cancer agents.

Elevated IRF9 raised cell apoptosis and tissue damages through suppressing SIRT1 in hyperlipidemia acute pancreatitis with liver injury

BackgroundHyperlipidemia is a vital etiology of acute pancreatitis (AP), 12 to 20% of which have a history of hyperlipidemia. Multiple organ failure is the major cause of the high mortality rate of AP. Liver injury has been discovered in 80% of AP patients. The relationship and role of IRF9 and SIRT1 have not been presented in AP and hyperlipidemia AP (HLAP) with liver injury. This investigation was designed to explore the function and relationship of IRF9 and SIRT1.MethodsHLAP model in vivo was performed by feeding high-fat forage and induced by peritoneal injection with 20% L-arginine. The severity of pancreas and liver tissues was assessed. Cell apoptosis in the liver was determined by the TUNEL experiment. IRF9, SIRT1, p53, and acetylated p53 (Ac-p53) expression levels in liver tissues were detected by qRT-PCR and Western blot. The association of IRF9 expression with SIRT1 levels was evaluated. The relevance of triglyceride level to tissue damage was analyzed.ResultsOur observation exhibited more distinct liver damage, a large number of hepatic cell apoptosis, marked raised IRF9, Ac-p53, and sharply dropped SIRT1 in the AP and HLAP groups. Compared with other groups, HLAP showed the most significant changes in liver injury, hepatic cell apoptosis, protein, and mRNA levels. The declined expression of SIRT1 was correlated with the elevated expression of IRF9. The damage of the pancreas and liver exacerbated with the increase in triglyceride levels.ConclusionElevated IRF9 in pancreatitis with liver injury raised cell apoptosis and tissue damage by decreasing SIRT1 expression.

Establishment and refinement of a DEN-induced hepatocellular carcinoma model in rats

Abstract Objectives Hepatocellular carcinoma is one of the most common malignant tumors in the world with complex etiology, high tumor heterogeneity, and low efficacy of treatment. The establishment of an animal model that is close to the clinical situation of hepatocellular carcinoma and can be successfully modeled many times is of great significance to the study of the pathogenesis, diagnosis and treatment of hepatocellular carcinoma. Methods We used Diethylnitrosamine (DEN) to induce hepatocellular carcinoma in rodents and compared four models of DEN-induced hepatocellular carcinoma. Group C (Control): rats were fed a standard laboratory rat diet and freely drank normal water. Group P (Peritoneal injection): rats were administered an IP injection (50 mg/kg/week) between 5 and 23 weeks after birth. Ten microliter of DEN solution would be injected per g of rat. Group O (DEN-Fed group): rats were allowed unrestricted access to water contaminated with 0.01 % DEN between the ages of 7 and 15 weeks. 0.2 mL of DEN drinking water was consumed per gram of rat. Group P+O (Combined peritoneal injection and Oral feeding): rats were administered an IP injection (50 mg/kg) at weeks 3 and 5 post-birth, and they freely drank water contaminated with 0.012 % DEN between weeks 7 and 14 post-birth. We used an ultrasound scan, biochemical testing, haematoxylin, and eosin staining, Masson staining, Wolf scarlet staining, Ki67, CD34, a-SMA, CD8, and CD68 staining to compare between groups. Results Liver dissection and ultrasound scan showed that compared to other groups, the liver of Group P+O was darker in color, with more grey-white cancer nodules and larger localized tumors, and the structure of the tumors was slightly disorganized, with the elastography hardness of the middle lobe and the right lobe was slightly increased. The alanine aminotransferase and total bilirubin of Group P+O were higher than those of Group O but lower than those of Group P. Haematoxylin and eosin staining showed that the tumors of Group P+O were large, with large tumor cords and pseudo-glandular, the degree of differentiation was medium and surrounded by more fatty lesions. Conclusions We conclude that combined DEN treatment is more effective, stable, and has the advantage of multiple modalities, leading to faster tumor formation.

Mesothelin-based CAR-T cells exhibit potent antitumor activity against ovarian cancer

BackgroundOvarian cancer (OC) is characterized by its rapid growth and spread which, accompanied by a low 5-year survival rate, necessitates the development of improved treatments. In ovarian cancer, the selective overexpression of Mucin-16 (MUC16, CA125) in tumor cells highlights its potential as a promising target for developing anti-tumor therapies. However, the potential effectiveness of CAR-T cell therapy that targets MUC16 in ovarian cancer cells is unknown.MethodsThe expression of MUC16 in viable OC cells was detected using immunofluorescence and flow cytometry techniques. A MSLN-CAR construct, comprising the MUC16-binding polypeptide region of mesothelin (MSLN), a CD8 hinge spacer and transmembrane domain, 4-1BB, and CD3ζ endo-domains; was synthesized and introduced into T cells using lentiviral particles. The cytotoxicity of the resultant CAR-T cells was evaluated in vitro using luciferase assays. Cytokine release by CAR-T cells was measured using enzyme-linked immunosorbent assays. The anti-tumor efficacy of the CAR-T cells was subsequently assessed in mice through both systemic and local administration protocols.ResultsMSLN-CAR T cells exhibited potent cytotoxicity towards OVCAR3 cells and their stem-like cells that express high levels of MUC16. Also, MSLN-CAR T cells were inefficient at killing SKOV3 cells that express low levels of MUC16, but were potently cytotoxic to such cells overexpressing MUC16. Moreover, MSLN-CAR T cells delivered via tail vein or peritoneal injection could shrink OVCAR3 xenograft tumors in vivo, with sustained remission observed following peritoneal delivery of MSLN-CAR T cells.ConclusionsCollectively, these results suggested that MSLN-CAR T cells could potently eliminate MUC16- positive ovarian cancer tumor cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for MUC16-positive patients.

The effects of ethanolic extract of Punica granatum Peel on Pentylenetetrazole-induced seizures in mice

Introduction: Pomegranate (Punica granatum L) is an ancient and unique fruit used as antioxidant, antidiabetic, anti-obesity, and antihypertensive. Considering oxidative stress and its relationship with convulsions and the antioxidant properties of pomegranate extract, the present study aimed to investigate the effect of ethanolic extract of Punica granatum peel on pentylenetetrazole (PTZ)-induced seizures in mice. Method: In this experimental study, male rats were randomly divided into four groups (n=8 in each group, PTZ group, and three groups received 100, 200, and 400 mg/kg of the extract). Thirty minutes after peritoneal injection of different doses of the extract, PTZ (100 mg/kg) was injected, and the mice were immediately transferred to a special cage, and the seizure parameters were evaluated for 60 minutes. Results: The results implicated that treating with ethanolic extract of Punica granatum peel in different doses of 100, 200, and 400 mg/kg body weight could delay the onset of minimal clonic seizures (MCS) caused by PTZ injection. In addition, doses of 200 and 400 mg/kg delay the onset of generalized tonic-clonic seizures (GTCS) caused by PTZ injection (P<0.001 to P<0.05). The anti-seizure effects of the ethanolic extract of Punica granatum were dose-dependent; therefore, the high doses have the most prophylactic effects against MCS and GTCS seizures (P<0.05). Conclusion: The results of this study showed that treatment with ethanolic extract of Punica granatum peel has an anti-seizure effect on mice, and the highest anti-seizure effect was observed in the highest dosage

Mouse Melanoma Model in Tumor Vaccines and Immunotherapy Research.

Efficacy of novel cancer immunization protocols could be tested in cell line-derived xenograft tumor models (CDX), which are based on the implantation of human tumor cell lines into mice for the development of different tumors by numerous means, such as subcutaneous implantation and orthotopic, venial, or peritoneal injections. However, the disadvantages of this model are the biological alteration of the derived cells or the inability of the cell lines to accurately reflect the complexity of tumor heterogeneity. Therefore, syngeneic mouse models, which offer a relatively simple grafting technique, preservation of lineage hierarchy, and the ability to generate tumors in as little as 2-8weeks, are being used to study potential future applications in medical treatment, particularly immunotherapies. Here, we describe a B16.F10 C57Bl/6 mouse melanoma model we selected for therapeutic studies employing IL-2 and IL-12 immunization protocols. Procedure of tumor cells inoculation and melanoma development in mice is described in detail, as first and necessary set-up for successful immunization experiments.

Study of the Effect of Wild-Type and Transiently Expressing CXCR4 and IL-10 Mesenchymal Stromal Cells in a Mouse Model of Peritonitis.

Sepsis due to peritonitis is a process associated with an inflammatory state. Mesenchymal stromal cells (MSCs) modulate the immune system due to the paracrine factors released and may be a therapeutic alternative. Three treatment groups were developed in a murine model of peritonitis to verify the effect of human adipose mesenchymal stem cell (hASCs). Additionally, a temporary modification was carried out on them to improve their arrival in inflamed tissues (CXCR4), as well as their anti-inflammatory activity (IL-10). The capacity to reduce systemic inflammation was studied using a local application (peritoneal injection) as a treatment route. Comparisons involving the therapeutic effect of wild-type ASCs and ASCs transiently expressing CXCR4 and IL-10 were carried out with the aim of generating an improved anti-inflammatory response for sepsis in addition to standard antibiotic treatment. However, under the experimental conditions used in these studies, no differences were found between both groups with ASCs. The peritoneal administration of hASCs or genetically modified hASCs constitutes an efficient and safe therapy in our model of mouse peritonitis.

Evaluation of the Anti-Oxidative Effects of Monocyte Cells Treated with Bone Marrow Mesenchymal Stem Cells Supernatant on Experimental Colitis in BALB/c Mice

Background: Alternate activation of monocytes could induce anti-inflammatory impacts. This study aimed to investigate whether monocyte cells treated with bone marrow mesenchymal stem cells supernatant (MSC-Sp) could improve anti-inflammatory responses as a cell transfer therapy for colitis. Materials and Methods: The induction of experimental colitis was done by acetic acid in four groups of male BALB/c mice, including the control colitis, treated-monocytes, non-treated-monocytes, and mesalazine groups. Following MSCs culture, the supernatant was harvested, and then 50% conditioned media, or negative control media was added to the monocytes for 24 h. After ten days, peritoneal injection of treated or non-treated-monocytes (105 cells/100µL) was performed in animals' relevant groups of colitis. Ten days later, the oxidative stress profile and histopathological evaluation of colon tissue were assessed. Results: Treated monocytes showed a significant improvement in the oxidative stress profile, namely myeloperoxidase (0.126±0.008), nitric oxide (0.153±0.01), and malondialdehyde (0.148±0.014) compared to the control colitis group (P<0.05). Also, histopathological results revealed that the rate of damage in the treated-monocytes group was less than in normal mice. Conclusion: Our study indicated that the treated monocytes had anti-oxidative potential in colitis mice and were usable as a complementary therapy. [GMJ.2021;10:e2131]

Protective effect of metformin on pulmonary fibrosis caused by paraquat through activating AMP-activated protein kinase pathway

To observe whether metformin (MET) inhibits transforming growth factor-β1 (TGF-β1)/Smad3 signaling pathway by activating adenosine activated protein kinase (AMPK), so as to alleviate the pulmonary fibrosis caused by paraquat (PQ) poisoning in mice. Male C57BL/6J mice were randomly divided into the Control group, PQ poisoning model group (PQ group), MET intervention group (PQ+MET group), AMPK agonist group (PQ+AICAR group), and AMPK inhibitor group (PQ+MET+CC group), according to a random number table method. A mouse model of PQ poisoning was established by one-time peritoneal injection of 1 mL PQ solution (20 mg/kg). The Control group was injected with the same volume of normal saline. After 2 hours of modeling, the PQ+MET group was given 2 mL of 200 mg/kg MET solution by gavage, the PQ+AICAR group was given 2 mL of 200 mg/kg AICAR solution by intraperitoneal injection, the PQ+MET+CC group was given 2 mL of 200 mg/kg MET solution by gavage and then 1 mL complex C (CC) solution (20 mg/kg) was intraperitoneally injected, the Control group and PQ group were given 2 mL of normal saline by gavage. The intervention was given once a day for 21 consecutive days. The 21-day survival rate of ten mice in each group was calculated, and the lung tissues of remaining mice were collected at 21 days after modeling. The pathological changes of lung tissues were observed under light microscope after hematoxylin-eosin (HE) staining and Masson staining, and the degree of pulmonary fibrosis was evaluated by Ashcroft score. The content of hydroxyproline in lung tissue and oxidative stress indicators such as malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. The protein expressions of E-cadherin, α-smooth muscle actin (α-SMA), phosphorylated AMPK (p-AMPK), TGF-β1 and phosphorylated Smad3 (p-Smad3) in lung tissue were detected by Western blotting. Compared with the Control group, the 21 days survival rate was significantly reduced, lung fibrosis and Ashcroft score were significantly increased in PQ group. In addition, the content of hydroxyproline, MDA and the protein expressions of α-SMA, TGF-β1 and p-Smad3 in lung tissue were significantly increased, while the activity of SOD and the protein expressions of E-cadherin and p-AMPK were significantly decreased in PQ group. Compared with the PQ group, the 21 days survival rates of mice were significantly improved in the PQ+MET group and PQ+AICAR group (70%, 60% vs. 20%, both P < 0.05). The degree of pulmonary fibrosis and the Ashcroft score were significantly reduced (1.50±0.55, 2.00±0.63 vs. 6.67±0.52, both P < 0.05). The content of hydroxyproline and MDA in lung tissue, as well as α-SMA, TGF-β1 and p-Smad3 protein expressions were significantly reduced [hydroxyproline (mg/L): 2.03±0.11, 3.00±0.85 vs. 4.92±0.65, MDA (kU/g): 2.06±1.48, 2.10±1.80 vs. 4.06±1.33, α-SMA/GAPDH: 0.23±0.06, 0.16±0.06 vs. 1.00±0.09, TGF-β1/GAPDH: 0.28±0.03, 0.53±0.05 vs. 0.92±0.06 p-Smad3/GAPDH: 0.52±0.04, 0.69±0.06 vs. 1.11±0.10, all P < 0.05], SOD activity and the protein expressions of E-cadherin and p-AMPK were significantly increased [SOD (μmol/g): 39.76±1.35, 33.03±1.28 vs. 20.08±1.79, E-cadherin/GAPDH: 0.91±0.08, 0.72±0.08 vs. 0.26±0.04, p-AMPK/GAPDH: 0.62±0.04, 0.60±0.01 vs. 0.20±0.04, all P < 0.05]. However, these protective effects of MET were inhibited by the addition of AMPK inhibitor CC solution. MET can effectively alleviate the degree of pulmonary fibrosis in mice poisoned with PQ, and its mechanism may be related to the activation of AMPK and inhibition of TGF-β1/Smad3 signaling pathway, which can be inhibited by AMPK inhibitor CC.

Effects of acupuncture on RT-BM1 and maternal-fetal immune tolerance in rats with ovarian hyperstimulation: 针刺对超排卵大鼠RT-BM1及母胎免疫耐受的影响

ObjectiveThis study aimed to evaluate the effects of acupuncture on embryo implantation and maternal–fetal immune tolerance in rats with ovarian hyperstimulation, identify the mechanisms of acupuncture treatment, and promote application of acupuncture in the field of assisted reproduction. MethodsEighty female Wistar rats were randomized into normal (N), model (M), acupuncture treatment (A), and progesterone treatment (P) groups, with 20 rats in each group. An ovarian hyperstimulation model was established in groups M, A, and P by the peritoneal injection of pregnant mare serum gonadotropin (PMSG) combined with human chorionic gonadotropin (HCG). Bilateral “Sanyinjiao (SP6)”, “Zusanli (ST36)” and “Taichong (LR3)” as acupoints were needled in rats in group A. Rats in group P received an intramuscular injection of progesterone (4 mg/kg). All pregnant rats were treated for seven days. On the 8th day, the pregnant rats were sacrificed for testing. The expression levels of estrogen (E2) and progesterone (P4) in serum and their receptors in the endometrium, Th1 and Th2 cytokines in serum and endometrium, RT-BM1 (the counterpart of HLA-G in rats) mRNA, Ly49 activating receptors (Ly49s3, Ly49s5) and inhibitory receptors (Ly49i3, Ly49i4, Ly49i5) on immunocytes in the maternal–fetal interface were detected using ELISA, Western blot, and real-time polymerase chain reaction (PCR). ResultsCompared with group N, the pregnancy rate and the number of implanted embryos were significantly decreased in group M (P < 0.05); E2 and P4 levels in serum, and the protein levels of ER-α and Progesterone Receptor (PGR) in the implantation site of endometrial tissue were significantly reduced in group M (P < 0.05); the expression of INF-γ and IL-2 in serum and endometrium were significantly increased, while the expression of IL-4, IL-6, and IL-10 in serum and endometrium were significantly decreased in group M(P < 0.05). The mRNA levels of RT-BM1, Ly49i3, and Ly49i4 receptors in group M were significantly higher than those in group N, whereas the mRNA levels of Ly49i5 and Ly49s5 receptors in group M were significantly lower than those in group N at the maternal–fetal interface (P < 0.05). The pregnancy rate and number of implanted embryos were significantly higher in group A than in group M (P < 0.05). Serum E2 and P4 levels were significantly higher in groups A and P than in group M (P < 0.05). The expression of ER-α and PGR in the implantation site of endometrial tissue in group A were significantly higher than those in group M (P < 0.05). The serum levels of INF-γ and IL-2 in group A and group P were significantly lower than those in group M (P < 0.05). The serum expression of IL-4, IL-6, IL-10 in group A and group P were significantly higher than those in group M (P < 0.05). The endometrium expression of INF-γ and IL-2 in group A were significantly lower than those in group M (P < 0.05). The endometrium expression of IL-4, IL-6, IL-10 in group A were significantly higher than those in group M (P < 0.05). The endometrium expression of INF-γ in group P were significantly lower than those in group M (P < 0.05). The endometrium expression of IL-6 and IL-10 in group P were significantly higher than those in group M (P < 0.05). The mRNA levels of RT-BM1 and these Ly49 receptors (except the Ly49s3 receptor) at the maternal–fetal interface of groups A and P were higher than those of group M (P < 0.05). ConclusionAcupuncture can significantly increase the pregnancy rate and average number of implanted blastocysts in rats with ovarian hyperstimulation. This mechanism is related to the effects of acupuncture in upregulating the expression of RT-BM1, regulating the balance of Th1/Th2 cytokines, and upregulating the expression of inhibitory receptors in immunocytes.

Peptidyl Arginine Deiminase (PAD) 4 Modulates the GI Microbiome and Baseline Inflammation in Mice: Implications for Sepsis

Introduction: As part of the innate immune response, neutrophils release neutrophil extracellular traps (NETs), webs of decondensed chromatin coated with histones that capture bacteria but cause collateral tissue damage when produced in a dysregulated manner. Deficiency of PAD4, a protein that converts histone arginine to citrulline to promote chromatin decondensation essential to NET release (NETosis), has been associated with improved outcomes in murine sepsis models, leading some to propose that inhibiting NETosis may be protective. This conclusion has primarily been drawn from studies using the murine cecal-ligation and puncture (CLP) model of polymicrobial sepsis, in which the cecum is perforated to disseminate GI microbiota. However, NET release is an evolutionarily conserved function, present in protozoa and retained in all studied animal species, suggesting that it plays a crucial role in the immune response. In this study, we seek to characterize the baseline immunophenotype of NET-deficient, PAD4 knockout (PAD4 -/-) mice, to determine how PAD4 influences the GI microbiome to impact outcomes in GI models of polymicrobial sepsis. Methods: Whole blood samples were collected from wild type (WT) C57BL/6 mice, PAD4 -/- mice generated by our group through deletion of exon II in the PAD4 gene (PAD4D ΔII), and PAD4 -/- mice with deletion of exon IX and X (PAD4D ΔIX+X) obtained from Jackson laboratory. Complete blood counts (CBCs) and weights were obtained from PAD4 -/- and healthy WT mice at 9-12 weeks. The Olink target 96 mouse exploratory panel was used to quantify variation in levels of 96 plasma proteins. Next generation sequencing was used to assess the abundance of different bacterial species in stool samples from WT, PAD4D ΔII, and PAD4D ΔIX+X animals. Polymicrobial sepsis was then induced through peritoneal injections of cecal slurry (CS), using cecal samples from WT, PAD4D ΔII, and PAD4D ΔIX+X donor mice. 24 hours post injection, mean sepsis score (MSS) was assessed, and blood was obtained for CBC measurement, plasma proteomic analysis, and quantification of bacterial colony forming units (CFUs). Relative abundance of bacterial species was quantified in blood and liver homogenates. Survival outcome was performed on parallel subsets of WT and PAD4 -/- mice. Results: At comparable ages, both strains of PAD4 -/- animals had lower weights and significantly higher baseline neutrophil counts than WT controls bred in the same colony. Proteomic analysis revealed that PAD4D ΔII mice had baseline elevation in the inflammatory markers IL6 and Plin1, while PAD4D ΔIX+X mice, the strain with more pronounced neutrophilia, had higher GM-CSF and decreased IL23r, proteins crucial to neutrophil mobilization. In CS sepsis studies, WT animals that received WT CS were sicker with higher MSS, lower platelet counts, higher inflammatory markers, and a 30% mortality rate. In contrast, PAD4D ΔII mice that received PAD4D ΔII CS had a milder clinical course, lower bacterial dissemination, and 100% survival (Fig2). To assess the basis for this unexpected outcome, we compared bacterial colony-forming units (CFU) in WT to PAD4 -/- CS and found lower levels of bacteria in PAD4D ΔII CS. Deep sequencing of stool from WT, PAD4D ΔII, and PAD4D ΔIX+X animals revealed marked differences in the relative abundance of bacterial species. Sequencing of blood and liver samples obtained 24 hrs after CS injection showed that the predominant proliferating organism in WT mice was Enterococcaceae species while there was an abundance of Lactobacillaceae species in the blood and liver of PAD4D ΔII mice (Fig1). Sepsis studies repeated with genotype switched CS showed that WT mice treated with PAD4D ΔIX+X CS had a milder disease course with 100% survival, whereas PAD4D ΔIX+X mice injected with WT CS had elevated MSS, a trend towards higher plasma inflammatory markers, and a 50% mortality rate, similar to that observed in WT mice injected with WT CS (Fig2). Conclusions: These studies show that NETosis influences baseline inflammation and modulates the GI microbiome in mice. Differences in disease severity and survival between PAD4 -/- and WT mice observed in the CS model of polymicrobial sepsis is primarily due to greater pathogenicity of the WT CS inoculum rather than collateral tissue damage secondary to dysregulated NETosis. The basis for differences in inflammation and microbiome composition in PAD4 -/- mice needs to be examined.

Mulberry leaf multi-components exert hypoglycemic effects through regulation of the PI-3K/Akt insulin signaling pathway in type 2 diabetic rats

Ethnopharmacological relevancePhytochemicals have unique advantages in the treatment of diabetes due to their multi-target activity and low toxicity. Mulberry leaves, a traditional Chinese herbal medicine, have been used in the prevention and treatment of diabetes for centuries. The main active ingredients in mulberry leaves with regards to the hypoglycemic effect are 1-deoxynojirimycin, flavonoids, and polysaccharides. However, the combined hypoglycemic effects and mechanisms of mulberry leaf multi-components remain unclear. Aim of the studyThis study explored the anti-diabetic effects of mulberry leaf multi-components (MMC) and the role of the PI-3K/Akt insulin signalling pathway in improving insulin resistance. Materials and methodsThe main chemical components of MMC were analyzed using the phenol-sulfuric acid method, aluminum nitrate-sodium nitrite method, and HPLC-ultraviolet/fluorescence detection method. The T2DM rat model was created via feeding a high-fat diet and peritoneal injection of streptozotocin. T2DM rats were divided into four groups: model, model plus metformin, model plus low-dose, and model plus high-dose MMC groups (100 and 200 mg/kg body weight/day, respectively), and plus normal group for a total of five groups. MMC was administered by oral gavage for six weeks. Fasting blood glucose and serum lipid profiles were measured using a glucometer and an automatic biochemistry analyzer, respectively. Serum insulin and adipocytokine levels were analyzed by ELISA. Hepatic glucose metabolizing enzyme activity was evaluated by ELISA and the double antibody sandwich method. Expression of PI-3K/Akt signalling pathway proteins was analyzed by RT-PCR and Western blotting. ResultsExtracted 1-deoxynojirimycin, flavonoid, and polysaccharide purity was 70.40%, 52.34%, and 32.60%, respectively. These components were then mixed at a ratio of 1:6:8 to form MMC. MMC significantly reduced serum glucose, insulin, and lipid levels. In diabetic rats, MMC enhanced insulin sensitivity and alleviated inflammatory and oxidative damage by lowing adipocytokine levels and increasing anti-oxidative enzyme activity. Insulin resistance was also mitigated. MMC regulated the activity of key downstream enzymes of hepatic glucose metabolism via activating the expression of PI-3K, Akt, PDX-1, and GLUT4 at the mRNA and protein levels, thereby correcting hepatic glucolipid metabolism disorders and exerting a hypoglycemic effect. ConclusionMMC ameliorated hepatic glucolipid metabolism disorders and improved insulin resistance in T2DM rats by activating the PI-3K/Akt signaling pathway. These results highlight the multi-component, multi-target, and combined effects of MMC, and suggest it may be further developed as a hypoglycemic drug.

Effects of the combination of chitosan and Acinetobacter KU011TH on the growth and health performances and disease resistance of juvenile hybrid catfish (Clarias gariepinus × C. macrocephalus)

Aquatic animal health management has become a crucial component in the goal of increasing catfish aquaculture productivity. Additionally, hybrid catfish (Clarias gariepinus × C. macrocephalus) has been promoted as a highly profitable freshwater fish in Asia. Interestingly, the crucial diseases induced by Aeromonas hydrophila have been reported to greatly impede catfish production. To overcome this challenge, the aim was to investigate the effects of the oral administration of potentially synbiotic chitosan (CH) and Acinetobacter KU011TH (AK) on the growth performance, immunological responses, and disease resistance of hybrid catfish against A. hydrophila. The control group was fed a basal diet (A), the diet fed to treatment group B was supplemented with 20 mL of CH/kg diet (B), and the experimental feed fed to groups C-D was mixed with 1 × 108, 1 × 109 and 1 × 1010 CFU/mL AK coated with 20 mL of CH/kg diet. Five different groups of juvenile hybrid catfish were continuously fed the 5 formulated feeds for 4 weeks. The results revealed that all tested feeds did not significantly enhance the hybrid catfish's average daily gain, specific growth rate, feed conversion ratio, hematocrit and erythrocyte counts. Interestingly, the application of CH and AK significantly increased the leukocyte counts, respiratory burst, lysozyme activity, alternative complement pathway hemolytic activity, and bactericidal activity (P < 0.05). The expression levels of the immune-related genes in the whole blood, head kidney, and spleen were significantly increased after CH-AK application (P < 0.05), but this finding was not observed in the liver (P > 0.05). Additionally, after 14 days of A. hydrophila peritoneal injection, the fish in group C showed significantly higher survival rates of approximately 70.0 % compared with the control fish in groups B, D, and E (52.5 %, 40.0 %, 45.0 %, and 45.0 %, respectively) (P < 0.05). These results collectively suggest that short-term application of the diet fed to group C effectively boosted the immune responses and disease resistance of hybrid catfish against A. hydrophila.

A study of Alfa Feto Protein parameter in Serum and Histological Effects of Pups Liver by SEM in Pregnant Rats Exposed to Overdose of Aminoglycoside antibiotics

The study has been conducted in Animal House/Faculty of Sciences/University of Kufa between December 2022 and February 2022, fifteen female Albino Rats are used. Gentamicin is aminoglycoside antibiotics widely used during pregnancy for treatment the infections. The present study has been intended to show Alfa Feto Protein parameter in serum and the histological effects of gentamicin on pups liver by SEM in female Albino Rats. The females Rats are randomly divided into three main groups, comprising five rats for each group .The control group is given inta peritoneal injection of physiological normal saline and the second and third group are given inta peritoneal injection of gentamicin doses twenty, forty mg/kg/day respectively for 20 days from the first day to the end of experimental allocated for each female.The rats are sacrificed in 20 th (dpc) to study the Alfa Feto Protein parameter in serum and histological effects of gentamicin on pups liver. The physiological study show negative results in all study groups due to absent of malformations in pups and present histological effects in external architecture structure of pups liver compare with control groups. In conclusion; aminoglycoside gentamicin caused histological effects on pupa liver and not absent of malformations in pups due to negative results of Alfa Feto Protein parameter.

Early Molecular Immune Responses of Turbot (Scophthalmus maximus L.) Following Infection with Aeromonas salmonicida subsp. salmonicida.

Turbot aquaculture production is an important economic activity in several countries around the world; nonetheless, the incidence of diseases, such furunculosis, caused by the etiological agent A. salmonicida subsp. salmonicida, is responsible for important losses to this industry worldwide. Given this perspective, this study aimed to evaluate early immune responses in turbot (S. maximus L.) following infection with A. salmonicida subsp. salmonicida. For this, 72 fish were individually weighed and randomly distributed into 6 tanks in a circulating seawater system. For the bacterial challenge, half of the individuals (3 tanks with 36 individuals) were infected using a peritoneal injection with the bacterial suspension, while the other half of individuals were injected with PBS and kept as a control group. Several factors linked to the innate immune response were studied, including not only haematological (white blood cells, red blood cells, haematocrit, haemoglobin, mean corpuscular volume, mean cell haemoglobin, mean corpuscular haemoglobin concentration, neutrophils, monocytes, lymphocytes, thrombocytes) and oxidative stress parameters, but also the analyses of the expression of 13 key immune-related genes (tnf-α, il-1β, il-8, pparα-1, acox1, tgf-β1, nf-kB p65, srebp-1, il-10, c3, cpt1a, pcna, il-22). No significant differences were recorded in blood or innate humoral parameters (lysozyme, anti-protease, peroxidase) at the selected sampling points. There was neither any evidence of significant changes in the activity levels of the oxidative stress indicators (catalase, glutathione S-transferase, lipid peroxidation, superoxide dismutase). In contrast, pro-inflammatory (tnf-α, il-1β), anti-inflammatory (il-10), and innate immune-related genes (c3) were up-regulated, while another gene linked with the lipid metabolism (acox1) was down-regulated. The results showed new insights about early responses of turbot following infection with A. salmonicida subsp. salmonicida.