Intraperitoneal Research Articles

Zi Shen Wan Fang repaired blood–brain barrier integrity in diabetic cognitive impairment mice via preventing cerebrovascular cells senescence

BackgroundBlood–brain barrier (BBB) integrity disruption is a key pathological link of diabetes-induced cognitive impairment (DCI), but the detailed mechanism of how the diabetic environment induces BBB integrity disruption is not fully understood. Our previous study found that Zi Shen Wan Fang (ZSWF), an optimized prescription consisting of Anemarrhenae Rhizoma (Anemarrhena asphodeloides Bge.), Phellodendri Chinensis Cortex (Phellodendron chinense Schneid.) and Cistanches Herba (Cistanche deserticola Y.C.Ma) has excellent efficacy in alleviating DCI, however, whether its mechanism is related to repairing BBB integrity remains unclear. This study aims to reveal the mechanism of BBB integrity destruction in DCI mice, and to elucidate the mechanism by which ZSWF repairs BBB integrity and improves cognitive function in DCI mice.MethodsDiabetic mouse model was established by feeding a 60% high-fat diet combined with a single intraperitoneal injection of 120 mg/kg streptozotocin (STZ). DCI mice were screened with morris water maze (MWM) after 8 weeks of sustained hyperglycemic stimulation. ZSWF was administered daily at doses of 9.36 and 18.72 g/kg for 8 weeks. Cognitive function was evaluated using MWM, blood–brain-barrier (BBB) integrity was tested using immunostaining and western blot, the underlying mechanisms were explored using single-cell RNA sequencing (scRNA-seq), validation experiments were performed with immunofluorescence analysis, and the potential active ingredients of ZSWF against cerebrovascular senescence were predicted using molecular docking. Moreover, cerebral microvascular endothelial cells were cultured, and the effects of mangiferin on the expression of p21 and Vcam1 were investigated by immunofluorescence staining and RT-qPCR.ResultsZSWF treatment significantly ameliorated cognitive function and repaired BBB integrity in DCI mice. Using scRNA-seq, we identified 14 brain cell types. In BBB constituent cells (endothelial cells and pericytes), we found that Cdkn1a and senescence-associated secretory phenotype (SASP) genes were significantly overexpressed in DCI mice, while ZSWF intervention significantly inhibited the expression of Cdkn1a and SASP genes in cerebrovascular cells of DCI mice. Moreover, we also found that the communication between brain endothelial cells and pericytes was decreased in DCI mice, while ZSWF significantly increased the communication between them, especially the expression of PDGFRβ in pericytes. Molecular docking results showed that mangiferin, the blood component of ZSWF, had a stronger affinity with the upstream proteins of p21. In vitro experiments showed that high glucose significantly increased the expression of p21 and Vcam1 in bEnd.3 cells, while mangiferin significantly inhibited the expression of p21 and Vcam1 induced by high glucose.ConclusionOur study reveals that ZSWF can ameliorate cognitive function in DCI mice by repairing BBB integrity, and the specific mechanism of which may be related to preventing cerebrovascular cells senescence, and mangiferin is its key active ingredient.Graphical

Preceding sevoflurane inhalation in rats reduces the stress of intraperitoneal injection both in inexperienced experimenters and subject rats.

In this study, we found that sevoflurane inhalation in rats prior to intraperitoneal injection decreased stress in both inexperienced experimenters and subject rats. Inexperienced experimenters anaesthetised male Sprague Dawley rats by intraperitoneal injection of anaesthetics, with or without preceding sevoflurane inhalation. Analysis using a visual analogue scale indicated that participating experimenters felt less stress when they performed intraperitoneal injections on rats after subjecting them to sevoflurane inhalation than when performing intraperitoneal injections alone. Plasma corticosterone concentrations of the anaesthetised subject rats were lower when the intraperitoneal injection was preceded by sevoflurane inhalation. Thus, our results demonstrated that it is advisable for inexperienced experimenters, until they gain confidence with experience, to perform sevoflurane inhalation on male rats before intraperitoneal injections.

Exercise preconditioning and resveratrol reduce the susceptibility of rats with obstructive jaundice to endotoxin and alleviate lung injury

IntroductionEndotoxemia is a common issue for patients with biliary obstruction. The lung is the most affected organ by endotoxins. Exercise training can alleviate lipopolysaccharide (LPS)-induced lung inflammation and resveratrol has biological effects similar to exercise. In this study, we evaluated the protective effects of exercise preconditioning, resveratrol, and their combination on LPS-induced lung injury and mortality in rats with obstructive jaundice.MethodsEndotoxemia was simulated in rats by common bile duct ligation (CBDL) and intraperitoneal injection of low-dose LPS. The treatment groups were pretreated with exercise and/or resveratrol to assess their effects on lung injury and mortality. Immunohistochemistry, immunofluorescence, and ELISA were subsequently used to evaluate the impact of exercise and/or resveratrol on inflammation in lung tissue and bronchoalveolar lavage fluid.ResultsWe found that even in the early stages, compared to sham-LPS rats, low-dose LPS induced excessive systemic inflammatory responses in CBDL rats, as evidenced by a significant increase in TNF-α and IL-6, severe lung inflammation, lung injury, and higher mortality rates, indicating that cholestasis increased rats’ susceptibility to endotoxins. Exercise training reduced neutrophil infiltration in the lungs of model rats and IL-6 levels in bronchoalveolar lavage fluid. Both exercise training and resveratrol exhibited synergistic effects in reducing macrophage accumulation in lung tissues, lowering TNF-α and IL-6 levels in the lungs, and decreasing TNF-α concentration in bronchoalveolar lavage fluid. Additionally, exercise and combined interventions both significantly increased the expression of IL-10. The interventions induced a marked improvement in lung tissue pathological damage and lung edema in model rats and prolonged the survival time of rats with obstructive jaundice.DiscussionThis study demonstrates that exercise preconditioning and/or resveratrol can significantly reduce rats’ susceptibility to endotoxins after CBDL and alleviate lung injuries through their anti-inflammatory effects, thereby decreasing the mortality risk.

Bexarotene ameliorated the pulmonary inflammation and M1 polarization of alveolar macrophages induced by cigarette smoke via PPARγ/HO-1

BackgroundAlveolar macrophages (AMs) modulate pulmonary inflammation in chronic obstructive pulmonary disease (COPD), contributing to its progression. The PPARγ/RXRα heterodimer influences AM polarization induced by cigarette smoke (CS). Although PPARγ agonists suppress CS-induced M1 macrophage polarization, the impact of RXRα agonists on this process has not been determined. This study explored the effects and mechanisms of the RXRα agonist bexarotene on macrophage polarization in a COPD mouse model.MethodsC57BL/6 mice were assigned to the control, model, bexarotene, or model + bexarotene group. The COPD model was induced by CS exposure and intraperitoneal injection of cigarette smoke extract (CSE), followed by intraperitoneal administration of bexarotene. Additionally, MH-S cells were exposed to CSE and bexarotene. Lung tissues were subjected to hematoxylin-eosin staining, and emphysema and inflammatory scores were assessed. Cytokine levels and cell differentials in bronchoalveolar lavage fluid were measured, and macrophage polarization was evaluated using immunohistochemistry, flow cytometry, and qPCR.ResultsBexarotene effectively reduced inflammatory scores, cytokine levels, and neutrophil counts and ameliorated emphysema and M1 polarization of AMs in COPD model mice. Furthermore, while CSE exposure reduced PPARγ expression and the transcriptional activity of AMs, bexarotene enhanced the transcriptional response of PPARγ to CSE. HO-1 was identified as a potential target of PPARγ; its levels were assessed in AMs, revealing that bexarotene mitigated the CSE-induced reduction in HO-1. Notably, the effect of bexarotene was partially inhibited by the PPARγ inhibitor.ConclusionsOur results indicated that bexarotene may curb inflammation and M1 polarization in COPD through activation of the PPARγ/HO-1 pathway.

Sex differences in mitochondrial gene expression during viral myocarditis

BackgroundMyocarditis is an inflammation of the heart muscle most often caused by viral infections. Sex differences in the immune response during myocarditis have been well described but upstream mechanisms in the heart that might influence sex differences in disease are not completely understood.MethodsMale and female BALB/c wild type mice received an intraperitoneal injection of heart-passaged coxsackievirus B3 (CVB3) or vehicle control. Bulk-tissue RNA-sequencing was conducted to better understand sex differences in CVB3 myocarditis. We performed enrichment analysis and functional validation to understand sex differences in the transcriptional landscape of myocarditis and identify factors that might drive sex differences in myocarditis.ResultsAs expected, the hearts of male and female mice with myocarditis were significantly enriched for pathways related to an innate and adaptive immune response compared to uninfected controls. Unique to this study, we found that males were enriched for inflammatory pathways and gene changes that suggested worse mitochondrial electron transport function while females were enriched for pathways related to mitochondrial homeostasis. Mitochondria isolated from the heart of males were confirmed to have worse mitochondrial respiration than females during myocarditis. Unbiased TRANSFAC analysis identified estrogen-related receptor alpha (ERRα) as a transcription factor that may mediate sex differences in mitochondrial function during myocarditis. Transcript and protein levels of ERRα were confirmed as elevated in females with myocarditis compared to males. Differential binding analysis from chromatin immunoprecipitation (ChIP) sequencing confirmed that ERRα bound highly to select predicted respiratory chain genes in females more than males during myocarditis.ConclusionsFemales with viral myocarditis regulate mitochondrial homeostasis by upregulating master regulators of mitochondrial transcription including ERRα.

Evaluation of the effect of melatonin treatment on telomere length of the retinal pigment epithelium in streptozotocin-induced diabetic rat model

ObjectivesWe aimed to investigate the effect of diabetic retinopathy and melatonin treatment on the relative telomer lengths (RTL) in retinal pigment epithelium (RPE) cells in a streptozotocin-induced diabetic rat model.BackgroundTL can be used to evaluate diabetes mellitus, its complications, and the effectiveness of its treatment. However, TL assessment has not been performed in retinal cells in a diabetic retinopathy model until now.MethodsForty Sprague-Dawley male rats were randomly divided into four groups. The experimental groups were: Control Group (C): non- diabetic rats; Diabetes Mellitus Group (DM): rats induced to diabetes without treatment; Melatonin and Diabetes Mellitus Group (Mel + DM): rats induced to diabetes and after confirmation, treated with melatonin; Melatonin Group (Mel): rats were not induced to diabetes, treated with melatonin. Diabetes was induced by intraperitoneal administration of streptozotocin solution after 12 h food fasting. For eight weeks after the diabetes was induced, melatonin was administered via subcutaneous injection at a dose of 10 mg / kg. RTLs were measured by qPCR method with modifications. The comparison of averaged data among groups was performed using least significant difference (LSD) and Kruskal – Wallis Test and One way ANOVA test.ResultsRTL was significantly similar in control and melatonin group. RTL was thinnest in DM group, in addition melatonin treatment significantly prevented the RTL shortening in DM + Mel group (p = 0.031).ConclusionWe demonstrated that diabetic retinopathy led to the shortening of RTL in RPE cells in rats and melatonin treatment prevents this shortening.

Potential Effect of Defatted Mealworm Hydrolysate on Muscle Protein Synthesis in C2C12 Cells and Rats

(1) Background: the objective of this study was to examine the impact of defatted mealworm hydrolysate (DMH), formulated through protein hydrolysis, on muscle protein synthesis in C2C12 cells and rats; (2) Methods: C2C12 cells were treated with dexamethasone and DMH, and cell viability was quantified using the MTT assay. Twenty-four Sprague–Dawley rats were divided into three groups (control, DEX, and DEX + DMH) and treated for 8 weeks. The DEX and DEX + DMH groups were administered intraperitoneal injections of DEX at a concentration of 2.25 mg/kg over a 3-d period. The control and DEX groups were fed a control diet, whereas the DMH group had part of the protein composition of the control diet replaced with 3.5% of DMH. The impact of DMH on muscle protein synthesis was evaluated through the measurement of grip strength, gastrocnemius and tibialis anterior muscle weights, and the investigation of muscle protein synthesis and degradation factor mRNA expression utilising the real-time PCR method; (3) Results: in vitro experiments demonstrated that treatment with DMH at concentrations greater than 5 mg/mL markedly alleviated DEX-induced injury in C2C12 cells. In vivo experiments demonstrated that the mRNA expression levels of myogenin and myoblast determination proteins, which promote muscle protein synthesis, were significantly increased. Furthermore, rats fed DMH exhibited significantly enhanced grip strength and tibialis anterior weight; (4) Conclusions: these findings indicate that DMH may serve as a functional material capable of promoting muscle protein synthesis and that the utilization of proteolytic enzymes is advantageous for the effective utilization of mealworms.

Limonin Exhibits Anti-Inflammatory Effects by Inhibiting mTORC1 and Mitochondrial Reactive Oxygen Species in Psoriatic-like Skin Inflammation

Psoriasis is a chronic inflammatory skin disorder characterized by abnormal immune responses and keratinocyte hyperproliferation. Limonin, a bioactive compound found in citrus fruits, has anti-inflammatory properties in various models; however, its effects on psoriasis are not fully understood. We investigated the therapeutic potential of limonin in a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced psoriasis mouse model. Mice were treated with TPA to induce psoriasis-like skin lesions, followed by intraperitoneal administration of limonin (200 or 400 μg/mouse) for six days. The results showed that limonin improved psoriasis-related symptoms in a psoriasis-like mouse model by suppressing the mRNA expression of pro-inflammatory cytokines and inflammation-related antimicrobial peptides and regulating the expansion of myeloid cells and T cells. Specifically, limonin reduced glucose uptake and oxidative phosphorylation to shift the metabolic program in the inflamed skin cells of psoriasis-like mice. Limonin activates AMPK and proteins related to mTOR inhibition, thereby suppressing the mTOR signaling pathway. It also inhibits mitochondrial mass and mitochondrial ROS production, thereby preventing the development of dysfunctional mitochondria in inflamed skin cells. Overall, limonin modulates key immune responses and metabolic pathways related to inflammation and mitochondrial health in psoriasis. Therefore, it is a promising natural candidate for the treatment of psoriasis and various inflammatory skin diseases.

Inhibitory Effect of Oxibiol on the Process of Protein Modification by Water-Soluble Products of Photo-Oxidative Destruction of Bisretinoid A2E

It has previously been shown that heteroaromatic antioxidant oxibiol (N-acetylcysteinate 6-hydroxy-2-aminobenzothiazole) inhibits the process of fructosylation of serum albumin. The aim of this study is to elucidate the inhibitory effect of oxibiol on modification of proteins by photodegradation products of bisretinoid A2E, the main fluorophore of lipofuscin granules in retinal pigment epithelial cells of the human eye. It was shown that unlike a water-soluble fraction fraction from non-irradiated A2E-liposomes, a water-soluble fraction formed after irradiation of A2E-liposomes with visible light significantly modified albumin in a day after incubation at 37°C. Oxibiol in millimolar concentrations effectively inhibited this process. The inhibitory effect of oxybiol could be attributed to its antioxidant activity and the ability to compete with reactive aldehydes formed during the photooxidative degradation of A2E-cardiolipin liposomes. The acute toxicity of oxibiol in mice after intraperitoneal injection was studied, the values of LD 10 and LD 50 were determined. The results obtained demonstrate that oxibiol can be used in pharmacology for preventing and curing diseases associated with the development of oxidative stress in various fields of medicine, primarily in ophthalmology.

Natural small molecule hinokitone mitigates NASH fibrosis by targeting regulation of FXR-mediated hepatocyte apoptosis.

Liver fibrosis is the common fate of NASH and poses a major health threat with very limited pharmacological treatments. This study aims to investigate the preventive effect of hinokitone (HO), an isolated compound from Agathis dammara, on NASH fibrosis and its underlying mechanism. To investigate the effect of HO on NASH fibrosis, C57BL/6 mice were either fed a high-fat diet (HFD) in conjunction with intraperitoneal injection of CCl4 for 8 weeks or single CCl4 for 14 days to establish mouse liver fibrosis model, and HO was administered by gavage simultaneously. To elucidate the underlying mechanisms, HepG2 cells were stimulated by palmitic acid (PA) or tumor necrosis factor α plus actinomycin-D (Act-D + TNFα) to induce hepatocyte apoptosis model. Furthermore, hepatocyte Farnesoid-X-receptor (FXR) specifically knocked out mice were established by the albumin-Cre-loxP recombination enzyme system to ascertain the role of FXR in the anti-NASH fibrosis effects of HO. The results showed that HO presented dose-dependent anti-liver fibrosis efficacy in NASH mice induced by HFD + CCl4 and CCl4-induced mouse liver fibrosis. Cellularly, HO significantly inhibited PA-induced lipotoxic apoptosis and Act-D + TNFα-induced exogenous apoptosis in hepatocytes, which in turn prevented HSC activation. Mechanistically, bioinformatics analysis and surface plasmon resonance assay had identified hepatocyte FXR as a target of HO. Specifically, HO directly bound to FXR and upregulated its protein level by inhibiting proteasomal degradation. In turn, HO attenuated hepatocyte lipid deposition through upregulating the FXR's downstream target genes SHP and CES1, and reduced cleaved-CASP8 level, thereby inhibiting hepatocyte apoptosis. Furthermore, HO lost its function in the inhibition of hepatocyte apoptosis and liver fibrosis when knockout hepatocyte FXR. In conclusion, HO has an inhibitory effect on NASH fibrosis. This effect is mediated by targeting upregulation of hepatocyte FXR, which in turn attenuates hepatocyte apoptosis and thus indirectly inhibits the activation of HSCs.

Based on network pharmacology, the mechanism of Dioscin in alleviating renal tubular epithelial cell injury induced by calcium oxalate crystals was explored.

The commencement of kidney stone formation involves a crucial initial phase characterized by injury to renal tubular cells caused by calcium oxalate (CaOx). Dioscin (Dio) has been acknowledged for its potent anti-inflammation and anti-apoptotic properties; nevertheless, the impact and underlying Investigation into the molecular basis underlying the action of Dioscin in mitigating inflammation and apoptotic induced by exposure to calcium oxalate crystals in renal tissues remain unexplored.To comprehend the precise mechanism of Dioscin in the treatment of crystalline nephropathy, we conducted experiments utilizing a murine model of CaOx crystal deposition, induced by intraperitoneal administration of glyoxylate. An in vitro model was constructed using HK-2 cells exposed to calcium oxalate monohydrate (COM). To evaluate the effect of Dioscin on calcium oxalate crystal deposition by ROS assay, Western blotting, immunohistochemistry, Periodic Acid-Schiff staining (PAS) staining, hematoxylin-eosin (H&E) staining. Using network pharmacology and molecular docking methods, we explored the molecular mechanism of Dioscin in the treatment of CaOx-induced renal tubular epithelial cell injury. Subsequently, we conducted experiments to verify our findings.We observed a significant protective effect of Dioscin treatment against kidney oxidative stress and inflammation induced by CaOx. Then we predicted through network pharmacology that Dioscin exerts its anti-apoptotic effect through the NF-kappa B signaling pathway. Then we verified in vitro and in vivo that administration of Dioscin can alleviate the elevation of TLR4 and activation of the NF-kappa B signaling pathway induced by calcium oxalate, as well as attenuate renal apoptosis. Instead, the beneficial impact of this protection of Dioscin was reversed after overexpression of the TLR4.Dioscin has the potential to alleviate the activation of the NF-kappa B signaling pathway through TLR4, thereby exerting anti-inflammatory and anti-apoptotic effects. This study provides new ideas for the prevention and treatment of kidney stones.

Evaluation of Protective Antibody Response following Vaccination with Formaldehyde Inactivated Enterobacter cloacae

Background: Multi-drug-resistant (MDR) strains of Enterobacter cloacae are becoming increasingly prevalent worldwide, which is reducing treatment options. Objective: The purpose of the present study was to evaluate protective antibody response following vaccination with formaldehyde inactivated Enterobacter cloacae. Methodology: The study used formaldehyde inactivated MDR Enterobacter cloacae obtained from various clinical samples to immunize 10 Swiss albino mice through intradermal injection. After the third dose of immunization, the mice were challenged with live Enterobacter cloacae through intraperitoneal injection and were monitored for 14 days. Blood was collected from the tail 7 days after each booster and through cardiac puncture 14 days after the challenge. The serum antibody produced by the vaccine was evaluated for bactericidal activity and antigen-binding capacity using ELISA. Results: In this study, it was observed that 100% of the immunized mice survived after 14 days of challenge. The ELISA showed that all the immunized mouse sera had 100% bactericidal activity. Additionally, all the pre- and post-challenge immunized serum immunoglobulin G antibodies had significantly higher optical density values compared to the control mice in ELISA. Top of Form Conclusion: Our study showed that when formaldehyde inactivated MDR Enterobacter cloacae was used for intradermal immunization in Swiss albino mice, it produced antibodies that offered protection. Using a first-generation vaccine can provide a larger pool of antigens to elicit the desired immune response. Bangladesh Journal of Medical Microbiology, January 2024;18(1):44-49

ATP-Gated P2X7-Ion Channel on Kidney-Resident Natural Killer T Cells and Memory T Cells in Intrarenal Inflammation.

The P2X7 ion channel, a key sensor of sterile inflammation, has been implicated as a therapeutic target in glomerulonephritis, and P2X7-antagonistic nanobodies can attenuate experimental glomerulonephritis. However, little is known about the expression of P2X7 on renal immune cells. We used conventional immunofluorescence of kidney sections as well as intraperitoneal injection of nanobodies in mice followed by flow cytometry analysis of parenchymal T cells and RNA-sequencing to elucidate the expression and function of P2X7 on parenchymal and vascular immune cells in the mouse kidney. Our study showed that parenchymal T cells, including a large subset of natural killer T cells and CD69+ tissue-resident memory T cells, display much higher cell surface levels of P2X7 than vascular T cells. After a single intraperitoneal injection of P2X7-blocking nanobodies, P2X7 on parenchymal T cells was fully occupied by the injected nanobodies within 30 min. This resulted in an effective protection of these cells from NAD-induced cell death during cell preparation. Conversely, systemic injection of NAD that mimics sterile inflammation results in the selective depletion of P2X7hiCD69hi T cells from the kidney parenchyma. Our study uncovered a novel purinergic regulatory mechanism affecting kidney-resident T cell populations.

Relaxin-2 improves type I diabetes mellitus-induced erectile dysfunction in rats by protecting cavernous endothelial and smooth muscle function, and inhibiting penile fibrosis and apoptosis.

Diabetes mellitus-induced erectile dysfunction (DMED) responds poorly to first-line treatments, necessitating the development of new therapeutic strategies. Relaxin-2 (RLX-2) plays a crucial role in protecting vascular endothelium, vasodilatation, and antifibrosis in various diseases. However, its effects and mechanisms on DMED remain unclear. To investigate the effects and mechanisms of RLX-2 on DMED rats in vivo and vitro. For in vivo research, 30 Sprague-Dawley rats were allocated into three groups: control, DMED, and DMED + RLX-2. The induction of DMED in the rats was achieved through intraperitoneal administration of streptozotocin, with confirmation of ED status being conducted via the apomorphine test. Rats in the DMED + RLX-2 group received continuous RLX-2 treatment by osmotic pump. Following a 4-week treatment period, assessment of erectile function was carried out using cavernous manometry, and samples of corpus cavernosum tissues were procured for subsequent analysis. For in vitro research, human cardiac microvascular endothelial cells (HCMECs) were allocated into three groups: control, high glucose (HG, 40mM), and HG + RLX-2. HCMECs were cultured for 6 days and treated with RLX-2 for 48h before collection for subsequent experiments. In DMED rats, RLX-2 treatment partially improved erectile function. We observed relatively normalized functions of endothelial and smooth muscle cells with decreased levels of apoptosis and fibrosis in the penis. In vitro experiments also demonstrated the antihyperglycemic effects of RLX-2. RLX-2 can protect endothelial and smooth muscle function, and inhibit aberrant apoptosis and fibrosis in the corpus cavernosum, thereby improving erectile function in DMED rats. This may provide a novel treatment for DMED.

Chimeric antigen receptor macrophages targeting c-MET(CAR-M-c-MET) inhibit pancreatic cancer progression and improve cytotoxic chemotherapeutic efficacy

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors. Macrophages are abundant in the tumor microenvironment, making them an attractive target for therapeutic intervention. While current immunotherapies, including immune checkpoint inhibition (ICI) and chimeric antigen receptor T (CAR-T) cells, have shown limited efficacy in pancreatic cancer, a novel approach involving chimeric antigen receptor macrophages (CAR-M) has, although promising, not been explored in pancreatic cancer. In this study, we first investigated the role of CAR-M cells targeting c-MET in pancreatic cancer.MethodsThe effectiveness and rationality of c-MET as a target for CAR-M in pancreatic cancer were validated through bioinformatic analyses and immunohistochemical staining of samples from pancreatic cancer patients. We utilized flow cytometry and bioluminescence detection methods to demonstrate the specific binding and phagocytic killing effect of CAR-M on pancreatic cancer cells. Additionally, we observed the process of CAR-M engulfing pancreatic cancer cells using confocal microscopy and a long-term fluorescence live cell imaging system. In an in situ tumor model transplanted into NOD/SCID mice, we administered intraperitoneal injections of CAR-M to confirm its inhibitory function on pancreatic cancer. Furthermore, we validated these findings in human monocyte-derived macrophages (hMDM).ResultsBioinformatics and tumor tissue microarray analyses revealed significantly higher expression levels of c-MET in tumor tissues, compared to the paired peritumoral tissues, and higher c-MET expression correlated with worse patient survival. CAR-M cells were engineered using human monocytic THP-1 cell line and hMDM targeting c-MET (CAR-M-c-MET). The CAR-M-c-MET cells demonstrated highly specific binding to pancreatic cancer cells and exhibited more phagocytosis and killing abilities than the pro-inflammatory polarized control macrophages. In addition, CAR-M-c-MET cells synergized with various cytotoxic chemotherapeutic drugs. In a NOD/SCID murine model, intraperitoneally injected CAR-M-c-MET cells rapidly migrated to tumor tissue and substantially inhibited tumor growth, which did not lead to obvious side effects. Cytokine arrays and mRNA sequencing showed that CAR-M-c-MET produced higher levels of immune activators than control macrophages.ConclusionsThis study provides compelling evidence for the safety and efficacy of CAR-M therapy in treating pancreatic cancer. The results demonstrate that CAR-M-c-MET significantly suppresses pancreatic cancer progression and enhances the effectiveness of cytotoxic chemotherapy. Remarkably, no discernible side effects occur. Further clinical trials are warranted in human pancreatic cancer patients.

Flaxseed Linusorb Alleviates Collagen-Induced Rheumatoid Arthritis in Rats via Inhibiting the TLR4/NF-κb/MAPK Signal Pathway and Modulating Gut Microbiota.

Flaxseed linusorb (FL) has gradually garnered widespread attention in recent years because of its intriguing bioactivities like anti-inflammation, antimelanogenesis, and even anticancer effects. Based on its proven in vitro anti-inflammatory activity and mechanism, it is supposed that FL may also exhibit an in vivo effect in treating and preventing rheumatoid arthritis (RA). Collagen-induced arthritis (CIA) rat models were established to investigate the potential therapeutic effect of FL, which were intervened with FL via gavage (50 and 100 mg/kg B.W.) and intraperitoneal injection (10 and 20 mg/kg B.W.). After FL treatment, RA clinical symptoms were significantly alleviated, including reduced toe swelling volume and mitigated bone damage in CIA rats. Moreover, a decline in the expression of pro-inflammatory factors (i.e., TNF-α, IL-1β, and IL-6) and RA-related proteins (i.e., MMP-3, COX-2, and 5-LOX) was observed to effectively block the TLR4/NF-κB/MAPK signaling pathway. In addition, FL was discovered to modulate the diversity and composition of intestinal microbiota in CIA rats, where the level of g_Parvibacte, g_Allobaculum, g_Enterococcus, and unclassified_o_Lactobacillales could be significantly increased, whereas the level of Gram-negative bacteria g_Parabacteroides, g_Parasutterella, and g_Paludicola was notably reduced. In conclusion, FL shows promise in RA treatment by inhibiting inflammatory pathways and regulating the gut microbiota.

Effect of sodium butyrate on kidney and liver mitochondrial dysfunction in a lipopolysaccharide mousemodel.

Sodium butyrate can reduce inflammation, but it is not known if butyrate can improve mitochondrial dysfunction during sepsis. We tested butyrate to prevent or reverse lipopolysaccharide (LPS)-induced mitochondrial dysfunction in murine kidney and liver. C57BL/6 mice were grouped as control (n = 9), intraperitoneal (IP) LPS (n = 8), pretreatment with IP butyrate 600 (n = 3) or 1200 mg/kg (n = 8) followed 2 h later by LPS, posttreatment with IP butyrate 600 (n = 3) or 1200 mg/kg (n = 7) 1 h after LPS, or butyrate 1200 mg/kg only (n = 8). Kidney and liver tissue were collected at 24 h to measure mitochondrial respiration, electron transport system (ETS) complex activity and subunit expression, and content (citrate synthase [CS] activity and mtDNA/nDNA). Kidney mitochondrial respiration was decreased after LPS compared to controls. Pretreatment with butyrate 1200 mg/kg increased kidney OXPHOSCI+II, ETSCI+II, ETSCII, and CIV respiration compared to LPS; posttreatment did not achieve significant increases except for OXPHOSCI. Liver mitochondrial respiration exhibited a similar pattern as in kidney, but differences were not significant. ETS complex and CS activity did not differ between groups, but CI and CII subunit expression trended higher with butyrate in kidney. Changes in mtDNA/nDNA followed a similar pattern as respiration in kidney and liver with a decrease after LPS that was not present with butyrate pretreatment. These data show that butyrate can prevent-but not significantly reverse-the LPS-induced decrease in kidney mitochondrial respiration without a clear effect in liver. Mitochondrial protection was not attributable to changes in ETS complex activity but may reflect maintenance of ETS subunit expression.

Comparison of the Effect of Delay Phenomenon and Quercetin Application on the Viability of Dorsal Skin Island Flaps in Rats: An Experimental Study.

Surgical delay is any surgical intervention performed 7-14 days before flap elevation, separating part of flap from its vascular bed and aiming to decrease flap necrosis. However, delay surgery needs to be planned and performed as a separate surgical operation. Quercetin is a flavonoid with anti-inflammatory, and vasodilator effects. This study compares the effects of quercetin and surgical delay on flap survival. The study included 32 male Wistar rats divided into four groups: control group (group 1), surgical delay group (group 2), quercetin group (group 3), and both surgical delay and quercetin group (group 4). All dorsal skin island flaps were elevated based on deep circumflex iliac artery and 7 days were selected as waiting period after flap elevation, 50 mg/kg (0.5 mL) intraperitoneal quercetin administration period, and surgical delay period. Macroscopically flap necrosis rates were calculated and histopathological examination was performed to evaluate number of vessels, vessel lumen diameters, inflammation, epidermal damage, and dermal fibrosis scores. All rats were euthanized. Flap necrosis rates, inflammation, epidermal damage, and dermal fibrosis scores of group 3 and 4 were found to be lower than group 1 and 2 (P < 0.05). Vascular lumen diameter of group 2, 3, and 4 were found to be higher than group 1 (P < 0.05) but no statistically significant difference was found for this parameter between group 2, 3, and 4 (P > 0.05). The number of vessels were found to be higher in group 2, group 3, and group 4 compared with group 1, but this difference was not to be found statistically significant (P = 0.534). This study shows that quercetin application is more effective in reducing flap necrosis rates and anti-inflammatory effect than surgical delay and also has superior effect in terms of vasodilation.

SEX DIFFERENCES IN SYSTEMIC INFLAMMATORY RESPONSE IN LOW HYPOXIA-RESISTANT RATS

OBSERVATION. Individual differences in hypoxia tolerance and expression of the hypoxia-inducible factor, HIF-1α, were found in humans and laboratory animals. This allowed us to distinguish groups of animals with low and high resistance to oxygen deficiency. It was shown that low hypoxia-resistant animals are characterized by a more severe course of the systemic inflammatory response (SIR). Sex differences in the severity of SIRS in animals with low hypoxia tolerance have not been studied. OBJECTIVE To characterize morphological and molecular-biological features of lipopolysaccharide (LPS)-induced SIR in low hypoxia-resistant male and female Wistar rats. MATERIALS AND METHODS. Low hypoxia-resistant animals were distinguished by the time of “life” in a hypobaric barocamera at an altitude of 11500 m (less than 60 s). SWO was modeled by intraperitoneal injection of LPS at a dose of 1.5 mg/kg. One day later, morphologic changes in the lungs, liver, and thymus, as well as endotoxin levels, corticosterone, HIF1-α, and TGFβ concentrations in serum from low hypoxia-resistant female and male Wistar rats were evaluated. RESULTS. Differences were found in control groups in females and males of low hypoxia-resistant rats, characterized by higher HIF1-α, corticosterone and endotoxin levels in serum in females. Thus, in females 24 h after LPS injection the number of neutrophils in interalveolar septa of lungs was higher and acute accidental involution of thymus with narrowing of subcapsular zone without changes in volumetric fraction of cortical substance developed. Whereas in males widespread pathological changes in the liver and more pronounced manifestations of accidental involution accompanied by narrowing of the volume fraction of the thymus cortical substance were revealed. In the blood serum of females with SWO, the content of corticosterone increased, while in males the concentration of HIF1-α and the level of endotoxin increased, and the content of TGFβ decreased. CONCLUSIONS. Although female and male rats were characterized by low resistance to hypoxia, the course of LPS-induced SWO and the development of immune reactions in animals have sex differences.

Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations

Background:Mesenchymal stem cells (MSCs) derived from gestational tissues offer a promising avenue for prenatal intervention in congenital malformations although their application is hampered by concerns related to cellular plasticity and the need for invasive, high-risk surgical procedures. Here, we present naturally occurring exosomes (EXOs) isolated from amniotic fluid-derived MSCs (AF-MSCs) and their mimetic analogs (MIMs) as viable, reproducible, and stable alternatives. These nanovesicles present a minimally invasive therapeutic option, addressing the limitations of MSC-based treatments while retaining therapeutic efficacy.Methods:MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. A physicochemical, structural, and molecular comparison was conducted with exosomes (EXOs) released from the same batch of cells. Additionally, their distribution patterns in female mice were evaluated following in vivo administration, along with an assessment of their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs). The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake in two different cell types(fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams.Results:Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a threefold greater yield. Biodistribution patterns following intraperitoneal administration were comparable between the two particle types, with the uterus being among targeted organs. No toxic effects were observed in the dams during gestation, nor were there any malformations or significant differences in the number of viable versus dead fetuses detected. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo.Conclusions:The present data confirms the potential application of EXOs and MIMs as suitable tools for prevention and treatment of NTDs and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.