Rat Peripheral Blood Research Articles

Influence of neodymium-doped yttrium aluminum garnet laser exposure time on cytokine secretion in lipopolysaccharide-challenged rat peripheral blood mononuclear cells

Multiple investigators have suggested that infrared laser energy facilitates hard and soft tissue wound healing through various mechanisms, including the suppression of inflammation. This study investigated the influence of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser exposure time on pro-inflammatory cytokine/chemokine concentrations in lipopolysaccharide (LPS)-stimulated rat peripheral blood mononuclear cells (PBMCs). Cultured rat PBMCs were stimulated with various LPS concentrations (0, 10, 100, or 1000 ng/mL) and treated with Nd:YAG laser irradiation for 0 (control), 30, 45, or 60 s. For these experiments, the average power, pulse duration, and repetition rate remained constant at 5 W, 100 μs, and 20 Hz, respectively. Luminex magnetic microsphere immunoassays were used to compare the secretion of 27 inflammatory mediators from LPS-stimulated PBMCs in laser-irradiated versus control groups. Two-way analysis of variance was used to compare the main effects of laser exposure time and LPS concentration on cytokine/chemokine concentrations and evaluate the potential interaction between these factors. Four pro-inflammatory cytokines – tumor necrosis factor-α, macrophage inflammatory protein (MIP)-1α, MIP-2, and interferon gamma-induced protein-10 – exhibited a trend of reduced secretion in laser-irradiated cultures. The effect appeared more pronounced at longer exposure times (45 and 60 s). However, none of the 27 inflammatory mediators exhibited statistically significant reductions in concentrations in laser-irradiated cultures versus control cultures. These observations do not support a robust anti-inflammatory effect of Nd:YAG laser irradiation. Further studies should explore the potential impact of Nd:YAG laser irradiation on cytotoxicity and cellular growth kinetics and extend across a range of irradiation parameters and cell types.

Comparison the impact of mesenchymal stromal cells, their microvesicles and plasma enriched with soluble platelet factors on survival and apoptosis of rat spleen lymphocytes <i>in vitro</i>

BACKGROUND: The trophic function is one of the important but insufficiently studied features of mesenchymal stromal cells (MSCs), their microvesicles (MVs), and plasma enriched with soluble platelet factors (PRPs), which ensure the viability of target cells. AIM: To analyze the capability of MSCs, MVs, and PRP to affect the viability and spontaneous and activation-induced apoptosis of rat spleen lymphocytes during culturing in vitro. MATERIALS AND METHODS: MSCs were isolated from the mononuclear cell fraction of the femoral bone marrow of Wistar rats using the plastic adhesion method. MVs were obtained from the conditioned medium of MSCs by centrifugation at 14,500 g. PRP was prepared by freezing/thawing rat peripheral blood platelet concentrate. Rat spleen lymphocytes were isolated on a density gradient of 1.077 g/cm3. The viability and degree of lymphocyte apoptosis in vitro were assessed by flow cytometry for 7-AAD incorporation and binding to annexin V. RESULTS: The presence of MSCs at 10 and 20% concentrations caused an increase in the number of living intact and PMA-activated lymphocytes by the end of 3-day in vitro cultivation. Compared with controls, the amount of necrotic cells decreased 8.3–13.5 times, and the number of apoptotic cells decreased 2.3–4.0 times, mainly due to lymphocytes at the late stage of apoptosis. MVs of MSCs at the indicated concentrations did not significantly affect the viability of lymphocytes cultured in vitro but reduced the level of apoptosis of intact and PMA-activated lymphocytes by 3.6 (р=0.03) and 4.8–5.2 (р=0.048; р=0.03) times respectively. Studies have shown that 1.25% rat PRP has a growth-stimulatory activity against MSCs but not against lymphocytes cultured in vitro. In the culture of intact lymphocytes, PRPs did not significantly affect cell viability with a slight decrease (2.7–2.9 fold, p 0.05) in the number of necrotic cells. In the culture of PMA-activated lymphocytes, 1.25–2.50% PRP increased the number of living cells by 1.6–2.2 times (р=0.002; р=0.01) and the number of necrotic cells by two times (р=0.02). CONCLUSION: MSCs, MVs of MSCs, and PRP, in decreasing order, enhanced the viability of rat spleen lymphocytes and suppressed late apoptosis during in vitro cultivation. Lymphocytes activated by phorbol myristate acetate are more sensitive to their vital action compared with resting cells.

Pterocarya hupehensis Skan total flavones ameliorate rheumatoid arthritis in rats by suppressing formation of neutrophil extracellular traps

To investigate the therapeutic mechanism of Pterocarya hupehensis Skan total flavonoids (PHSTF) for rheumatoid arthritis (RA). Twenty-five male SD rats were randomly divided into normal control group, RA model group, PHSTF treatment (45 and 90 mg/kg) groups, and Tripterygium glycosides (TPG) tablet (10 mg/kg) group (n=5). Except for those in the normal control group, all the rats were subjected to collagen-induced arthritis (CIA) modeling using a secondary immunization method, after which PHSTF and TPG were administered via gavage once daily for 4 weeks. After the treatments, serum levels of TNF-α and IL-1β were measured using ELISA, and ankle joint pathologies were assessed with HE staining; the expression of citrullinated histone H3 (Cit-H3), a neutrophil extracellular trap (NET) marker, in the ankle joints was evaluated with immunohistochemistry. In primary cultures of rat peripheral blood neutrophils stimulated with phorbol ester (PMA), the effects of PHSTF (100 and 200 μg/mL) on the expressions of Cit-H3, peptidylarginine deiminase 4 (PADI4), neutrophil elastase (NE), and myeloperoxidase (MPO) were examined with Western blotting; immunofluorescence assay was used to observe Cit-H3 expression and NET formation in the cells. In the CIA rat models, PHSTF significantly alleviated ankle swelling, decreased serum levels of TNF-α and IL-1β, improved histopathological changes in the ankle joints, and reduced Cit-H3 expression in both the serum and ankle joint cartilage. In the isolated rat neutrophils, PHSTF showed no significant effect on cell viability but strongly inhibited PMA-induced NET release. PHSTF can alleviate RA by inhibiting the formation of NETs.

JAK1, SKI, ZBTB16 as potential biomarkers mediate the inflammatory response in keratoconjunctivitis sicca

Keratoconjunctivitis sicca (KCS) is an ocular condition characterized by insufficient tear production and inflammatory irritation, with Sjögren’s syndrome (SS) being a major causative factor. This study aimed to extract patient transcriptomic data from the GEO database to identify signature genes associated with the diagnosis and treatment of KCS and the expression of three key genes were experimentally verified.We performed a difference analysis on the SS patient dataset and performed a Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on the resulting genes. Additionally, a Weighted Gene Co-expression Network Analysis (WGCNA) was constructed. Machine learning techniques were employed to analyze the most strongly correlated gene modules with SS traits. These findings were further validated using KCS immune-correlation microarrays as a validation set. The correlation of the three identified genes with 22 immune cells was assessed through immune infiltration analysis. Subsequently, a rat model of desiccated keratoconjunctivitis was established, and the modeling situation and expression of characteristic genes were analyzed at the morphological, tissue, and molecular levels.Bioinformatic prediction revealed that the expression of JAK1, SKI, ZBTB16 not only differed in the machine learning validation set, but also correlated with some immune cells in the immune infiltration analysis. The results of animal experiments showed that the transcription and expression levels of these three genes were significantly different in rat KCS tissues and normal tissues, and there were also differences in the expression of JAK1 and SKI in rat peripheral blood, as well as significant up-regulation of the expression of related inflammatory factors in KCS tissues. Through bioinformatics prediction and animal experimental validation, this study identified three differentially expressed genes in SS mediated KCS patients, which provide new potential biological targets for the diagnosis and treatment of KCS.

Enhanced effect of limb remote ischemic postconditioning combined with paeoniflorin on alleviating cerebral ischemic injury via neutrophil NADPH pathway

BackgroundLimb remote ischemic postconditioning (LRIP) and paeoniflorin (PF) both can ameliorate cerebral ischemia reperfusion (I/R) injury. At present, whether LRIP combined with PF can achieve better therapeutic effect is unknown. PurposeThis study explored the alleviating effect and mechanism of LRIP in combination with PF on cerebral I/R injury in rats. MethodsMiddle cerebral artery occlusion (MCAO) surgery was performed on rats except Sham group. Then PF (2.5 mg/kg, 5 mg/kg, 10 mg/kg) was administrated by intraperitoneal injection 10 min before the start of reperfusion. LRIP was operated on the left femoral artery at 0 h of reperfusion. Behavioral testing was used to assess neurological impairment, while TTC staining was used to examine infarct volume. Protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox in neutrophils from rat peripheral blood were tested by Western blot. Rat bone marrow neutrophils were extracted and incubated for 24 h with serum from rats after LRIP combined with PF. p38 MAPK inhibitor group was administrated SB203580 while the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor group was administrated Apocynin. Neutrophils were stimulated by fMLP (10 μM). Reactive oxygen species (ROS) production and protein expression of MyD88, TRAF6, p38 MAPK, and p47phox (ser 304 and ser 345) were detected. ResultsLRIP combined with PF (5 mg/kg) reduced cerebral infarct volume, ameliorated neurological deficit score (NDS), decreased fMLP-stimulated ROS release and downregulated the protein expression of MyD88, TRAF6, p38-MAPK and phosphorylation of p47phox (ser 304 and ser 345) in neutrophils. ConclusionThe protective effect of LRIP combined with PF on cerebral I/R injury was better than either alone. Taken together, we provided solid evidence to demonstrate that the combination of LRIP and PF had potential to alleviate cerebral I/R injury, which was regulated by MyD88-TRAF6-p38 MAPK pathway and neutrophil NADPH oxidase pathway.

Osteogenic and angiogenic dual small molecule loaded chitosan nanoparticles for vascularized bone regeneration

AbstractIn recent years, the preference for small molecules has increasingly overshadowed growth factors. In this study, an osteogenic factor, DIPQUO, and an angiogenic factor, a VEGF inducer, GS4012, were encapsulated in chitosan nanoparticles (NPs) to provide vascularized bone regeneration. The encapsulation efficiencies of DIPQUO and GS4012 were 79% and 75%, respectively. The release profiles were 69% and 41% within the first 3 h, followed by 13 days to complete the release of all materials. The synthesis of NPs was validated via FT‐IR analysis, while their thermal properties were assessed via Differential scanning calorimetry (DSC). For in vitro osteogenesis, mesenchymal stem cells isolated from rat bone marrow were investigated by MTS, ALP, and various histological stains (H&E, alizarin red, and von Kossa). In the context of angiogenesis, endothelial cells derived from the peripheral blood of rats were isolated, characterized, and their capabilities to migrate and form vessels assessed. This study demonstrated that two different small molecules act synergistically to enable bone differentiation and may contribute to vascularized bone regeneration.

Induction effect of antiretroviral bictegravir on the expression of Abcb1, Abcg2 and Abcc1 genes associated with P-gp, BCRP and MRP1 transporters present in rat peripheral blood mononuclear cells

ABSTRACT Background Antiretrovirals have the potential to cause drug interactions leading to inefficacy or toxicity via induction of efflux transporters through nuclear receptors, altering drug concentrations at their target sites. Research Design and Methods This study used molecular dynamic simulations and qRT-PCR to investigate bictegravir’s interactions with nuclear receptors PXR and CAR, and its effects on efflux transporters (P-gp, BCRP, MRP1) in rat PBMCs. PBMC/plasma drug concentrations were measured using LC-MS/MS to assess the functional impact of transporter expression. Results Bictegravir significantly increased the expression of ABC transporters, with Car identified as a key mediator. This suggests that bictegravir’s influence on nuclear receptors could affect drug transport and efficacy at the cellular level. Conclusions Bictegravir activates nuclear receptors enhancing efflux transporter expression. Understanding these interactions is crucial for preventing drug–drug interactions and reducing toxicity in clinical use. Combining CAR antagonists with bictegravir may prevent drug resistance and toxicity. However, these findings are based on preclinical data and necessitate further clinical trials to confirm their applicability in clinical settings.

Chinese medicine Di-long (Pheretima vulgaris) and its active fraction exhibit anti-rheumatoid arthritis effects by inhibiting CXCL10/CXCR3 chemotaxis in synovium

Ethnopharmacological relevanceDi-Long (Pheretima vulgaris) is a classic animal sourced traditional Chinese medicine. It has been used for the treatment of joint inflammation and arthralgia for over two thousand years due to its effects of Tong-Luo-Zhi-Tong (dredging collaterals and alleviating pain). Our previous study showed that Chinese medicine Di-Long has significant anti-rheumatoid arthritis (RA) effects. Aim of the studyConsidering Di-Long as a potential source of active compounds with specific anti-RA therapeutic effects, this research was to obtain the anti-RA target-specific active fraction from Di-Long extracts (DL), and to further explore the chemical basis and verify the anti-RA mechanism of this active fraction. Materials and methodsTranscriptomic was applied to obtain the main anti-RA targets of DL on human RA fibroblast-like synoviocytes (FLS) and validated by qPCR. The target-corresponding active fraction was isolated from DL by ethanol precipitation and gel chromatography, and analyzed by nanoliter chromatography-mass spectrometry. Anti-RA effects of this active fraction was investigated by collagen-induced arthritis (CIA) in mice, and anti-RA mechanisms were verified in cocultured model of rat FLS and peripheral blood lymphocytes. ResultsWe confirmed that CXCL10/CXCR3 was the main anti-RA target of DL. The active fraction - A (2182 - 890 Da) was isolated from DL based on its CXCL10 inhibiting effects in RA-FLS. Fraction A contains 195 peptides (192 were newly discovered), 26 of which might be bioactive and were considered to be the chemical basis of its anti-RA effects. Fraction A significantly ameliorated the joint destruction and overall inflammation in CIA mice, and downregulated CXCR3 expression in mice joint. Fraction A inhibited the chemotaxis of Th-cells in rat peripheral blood lymphocytes towards the TNF-α-induced rat FLS through CXCL10/CXCR3 pathway. ConclusionsOur work indicated that active fraction from DL containing small peptides exhibits promising therapeutic effects for RA through inhibiting CXCL10/CXCR3 chemotaxis.

Optimization and verification of conditions for induction of neutrophil extracellular traps in vitro

This study aims to optimize the conditions for the formation of neutrophil extracellular traps(NETs) in vitro, so as to establish a relatively stable experimental research platform. Different conditions were compared, including commonly used laboratory animals(rats and mice) and a variety of cell sources(bone marrow neutrophils and peripheral blood neutrophils separated by percoll density gradient centrifugation). Different inducers like lipopolysaccharide(LPS) and phorbol 12-myristate 13-acetate(PMA) were used for induction in vitro. Myeloperoxidase(MPO)/citrullinated histone H3(CitH3)/DAPI immunofluorescence and cell free DNA(cf-DNA) content determination were used for comprehensive evaluation to screen the optimal conditions for the formation of NETs induced in vitro. Furthermore, the stability of the selected conditions for inducing the formation of NETs in vitro was evaluated by tetramethylpyrazine(TMP), an active component in Chinese herbal medicines. The results showed that coated poly-D-lysine(PDL) induced the formation of NETs in bone marrow neutrophils of mice to a certain extent. Both LPS and PMA significantly up-regulated the protein levels of MPO and CitH3 in mouse bone marrow neutrophils and elevated the cfDNA level in the supernatant of rat peripheral blood neutrophils. The cfDNA level in the PMA-induced group increased more significantly than that in the LPS-induced group(P<0.05). The results of immunofluorescence staining showed that the expression of MPO and CitH3 in mouse bone marrow neutrophils, rat bone marrow neutrophils, and rat peripheral blood neutrophils were significantly increased after PMA induction, especially in rat peripheral blood neutrophils. TMP significantly down-regulated the protein levels of MPO, CitH3, and neutrophil elastase(NE) in rat peripheral blood neutrophils induced by PMA. In conclusion, treating the peripheral blood neutrophils of rats with PMA is the optimal condition for inducing the formation of NETs in vitro. This study provides an optimal platform for in vitro studies based on NETs and a basis for studying the effects of traditional Chinese medicines targeting NETs.

Protective effect of thyroid and restores of ovarian function of Buzhong Yiqi granule on experimental autoimmune thyroiditis in female rats.

To observe the effects of Buzhong Yiqi granule on thyroid function and ovarian function in rats with experimental autoimmune thyroiditis (EAT). EAT model was replicate by using the method of mixing and injecting porcine thyroglobulin with Freund's adjuvant and high iodine. Rats were randomly divided into normal control (NC) group, EAT model (EAT) group, selenium yeast (PC) group, low dose Buzhong Yiqi (BZYQ-L) group, medium dose Buzhong Yiqi (BZYQ-M) group and high dose Buzhong Yiqi (BZYQ-H) group. After two months of drug intervention according to dosage, enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of free triiodothyronine (FT3), free thyroxine (FT4), thyroid-stimulating hormone (TSH), anti-thyroid peroxidase antibody (TPOAb), thyroglobulin antibody (TGAb) in peripheral blood of rats. The pathological changes of rat thyroid tissues were observed under light microscope with HE staining; ELISA was used to determine estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), anti-müllerian hormone (AMH), and the pathological changes of rat ovarian tissues were observed under light microscope with hematoxylin and eosin staining. Compared with the NC group, BZYQ granule improved the thyroid and ovarian tissue morphology, and the levels of TPOAb, TGAb and TSH in the model group rats significantly increased (P < 0.05), the thyroid tissue was severely destroyed, the levels of E2, FSH, LH, T, AMH significantly increased (P < 0.05), and the ovary exhibited polycystic changes; Compared with the model group, TSH level in the BZYQ-L group rats decreased (P < 0.05), FSH, T, AMH levels decreased (P < 0.05), in the BZYQ-M group TPOAb, TSH levels decreased (P < 0.05), FSH, LH, T, AMH levels significantly decreased (P < 0.05), BZYQ-H group TPOAb, TGAb, TSH levels significantly decreased (P < 0.05), FSH, LH, T, AMH levels significantly decreased (P < 0.05), with the greatest improvement and significantly better than selenium yeast group (P < 0.05). BZYQ granule could regulate the thyroid function of EAT rats, reduce thyroid antibody titers, then act on the ovarian function, regulate hormone disorders, and alleviate the pathological damage of rat's ovarian tissues. The effect of high dose Buzhong Yiqi granule is the best.

Gut microbial analysis combined with metabolomics reveal the mechanism of stachyose on blood deficiency syndrome in rats

Stachyose (ST), a naturally occurring compound extracted from cucumbers and legumes, holds great promise as a natural therapeutic agent. In this study, the impact of ST on blood deficiency syndrome (BDS) induced by cyclophosphamide (CP) and acetylphenylhydrazine (APH) was investigated in a rat model. Subsequently, the levels of erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), tumour necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) were quantified in rat blood and peripheral blood cells. Furthermore, haematoxylin and eosin (HE) staining was employed to elucidate morphological alterations in the spleen tissue. To gain comprehensive insights into the therapeutic potential and underlying mechanisms of ST against BDS, we used 16S rRNA gene sequencing and serum and spleen metabolomic analyses. Our findings revealed a significant ameliorative effect of ST on BDS. Notably, at the phylum level, Firmicutes and Bacteroidota exhibited marked variations in abundance, while at the genus level, discernible changes were observed in the abundances of Lactobacillus and Bifidobacterium, among others. The therapeutic effects of ST on BDS were proposed to be due to its modulation of phenylalanine metabolism and glycine, serine and threonine metabolism and its impact on the restructuring of the gut microbiota.

Transcriptome data mining combined with clinical and animal experiments for identification of syndrome element marker genes during entire course of steroid-induced osteonecrosis of femoral head

A research strategy combining transcriptome data mining and experimental verification was adopted to identify the marker genes characterizing the syndrome elements of phlegm, stasis, and deficiency in steroid-induced osteonecrosis of the femoral head(SONFH). Firstly, the common differentially expressed gene sets of SONFH with the syndromes of phlegm-stasis obstructing collaterals, vessel obstruction, and liver-kidney deficiency were obtained from the clinical transcriptomic analysis of a previous study. The differential expression trend analysis and functional gene mining were then employed to predict the candidate marker gene sets representing phlegm, stasis, and deficiency. The whole blood samples from SONFH patients, whole blood samples from SONFH rats, and affected femoral head tissue samples were collected for qPCR, which aimed to determine the expression levels of the candidate marker genes mentioned above. Furthermore, the receiver operating characteristic curve(ROC) was established to objectively evaluate the syndrome differentiation effectiveness of the candidate marker genes mentioned above. The transcriptome data analysis results showed that the candidate marker genes for phlegm was ELOVL fatty acid elongase 6(ELOVL6), and those for stasis were ankyrin 1(ANK1), glycophorin A/B(GYPA/B), and Rh-associated glycoprotein(RHAG). The candidate marker genes for deficiency were solute carrier family 2 member 1(SLC2A1) and stomatin(STOM). The qPCR results showed that compared with that in the non-SONFH group, ELOVL6 had the lowest expression level in the peripheral blood of the SONFH patients with the syndrome of phlegm-stasis obstructing collaterals(P&lt;0.05). Compared with that in the normal control group, ELOVL6 had the lowest expression level in the peripheral blood and affected femoral head tissue of SONFH rats modeled for 4 weeks(P&lt;0.01), and it showed better syndrome differentiation effectiveness of rats modeled for 4 weeks(AUC=0.850, P=0.006) than at other modeling time points(8, 12, 16, and 21 weeks, AUC of 0.689, 0.766, 0.588, and 0.662, respectively). Compared with that in the non-SONFH group, the expression levels of ANK1, GYPA, and RHAG were the lowest in the peripheral blood of SONFH patients with the vessel obstruction syndrome(P&lt;0.05). The expression levels of the three genes were the lowest in the peripheral blood and affected femoral head tissue of SONFH rats modeled for 12 weeks(P&lt;0.05, P&lt;0.01), and their syndrome differentiation effectiveness in the rats modeled for 12 weeks(GYPA: AUC=0.861, P=0.012; ANK1: AUC=0.855, P=0.006; RHAG: AUC=0.854, P=0.009) was superior to that for 4, 8, 16, and 21 weeks(GYPA: AUC=0.646, 0.573, 0.691, and 0.617, respectively; ANK: AUC1=0.630, 0.658, 0.657, and 0.585, respectively; RHAG: AUC=0.592, 0.511, 0.515, and 0.536, respectively). Compared with the non-SONFH group, both SLC2A1 and STOM had the lowest expression levels in the peripheral blood of patients with the syndrome of liver and kidney deficiency(P&lt;0.05). Compared with the normal control group, their expression levels were the lowest in the peripheral blood and affected femoral head tissue of SONFH rats modeled for 21 weeks(P&lt;0.05, except STOM in the peripheral blood of rats). Moreover, the syndrome differentiation effectiveness of SLC2A1 in the rats modeled for 21 weeks(AUC=0.806, P=0.009) was superior to that for 4, 8, 12, and 16 weeks(AUC=0.520, 0.580, 0.741, 0.774, respectively), and STOM was meaningless in syndrome differentiation. In summary, the candidate marker gene for phlegm in SONFH is ELOVL6; the candidate marker genes for stasis are GYPA, RHAG, and ANK1; the candidate marker gene for deficiency is SLC2A1. The results help to reveal the biological connotations of phlegm, stasis, and deficiency in SONFH at the genetic level.

Effect of peptides of trophoblastic Β1 glycoprotein on peripheral and local T-regulatory lymphocytes level in Wistar rats with allogeneous bone marrow cell transplantation

Objective. The range of peptide drugs is expanding, but no drug with immunosuppressive activity has yet been found. Considering the fact that the trophoblastic β1-glycoprotein (PSG) is a fetoplacental protein with immunosuppressive activity, short peptide fragments of this protein were studied in the formation of an immune response in a situation of allogeneic cell transplantation. To study the effect of PSG peptides (YECE, YQCE, YVCS, and YACS) on the levels of peripheral and local T-regulatory cells (Treg) during the formation of an immune response to the introduction of allogeneic bone marrow cells (BM) in a dynamic experiment on Wistar rats.&#x0D; Materials and methods. We used an original host-versus-graft model in male Wistar rats without preconditioning of recipients. Animals were injected with PSG peptide fragment composition against a background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment, in which the following parameters were evaluated: the level of peripheral "true" Tregs (CD4+CD25+FOXP3+), CD4+CD25-FOXP3+ cells, and FOXP3 expression in mesenteric lymph nodes. Material was collected on days 3 and 21 of the experiment.&#x0D; Results. PSG peptide administration against a background of allogeneic BM cells was found to reduce the absolute and relative amount of Treg in the peripheral blood of rats on days 3 and 21 of the experiment. PSG peptides against the background of the introduction of allogeneic BM cells reduced the absolute and relative amounts of CD4+CD25-FOXP3+ cells on the day 3 of the experiment. The introduction of PSG peptides against the background of the introduction of BM cells resulted in a relative decrease in FOXP3 expression in the T zone of mesenteric lymph nodes on the day 21 of the experiment.&#x0D; Conclusions. Thus, the PSG peptides did not have the expected effect on the level of peripheral and local Treg cells; moreover, the presence of the peptides led to a decrease in the number of these cells.

Clinical-grade human skin-derived ABCB5+ mesenchymal stromal cells exert anti-apoptotic and anti-inflammatory effects in vitro and modulate mRNA expression in a cisplatin-induced kidney injury murine model.

Acute kidney injury (AKI) is characterized by a rapid reduction in renal function and glomerular filtration rate (GFR). The broadly used anti-cancer chemotherapeutic agent cisplatin often induces AKI as an adverse drug side effect. Therapies targeted at the reversal of AKI and its potential progression to chronic kidney disease or end-stage renal disease are currently insufficiently effective. Mesenchymal stromal cells (MSCs) possess diverse immunomodulatory properties that confer upon them significant therapeutic potential for the treatment of diverse inflammatory disorders. Human dermal MSCs expressing ATP-Binding Cassette member B5 (ABCB5) have shown therapeutic efficacy in clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. In preclinical studies, ABCB5+ MSCs have also shown to reverse metabolic reprogramming in polycystic kidney cells, suggesting a capacity for this cell subset to improve also organ function in kidney diseases. Here, we aimed to explore the therapeutic capacity of ABCB5+ MSCs to improve renal function in a preclinical rat model of cisplatin-induced AKI. First, the anti-apoptotic and immunomodulatory capacity was compared against research-grade adipose stromal cells (ASCs). Then, cross-species immunomodulatory capacity was checked, testing first inhibition of mitogen-driven peripheral blood mononuclear cells and then modulation of macrophage function. Finally, therapeutic efficacy was evaluated in a cisplatin AKI model. First, ABCB5+ MSCs suppressed cisplatin-induced apoptosis of human conditionally-immortalized proximal tubular epithelial cells in vitro, most likely by reducing oxidative stress. Second, ABCB5+ MSCs inhibited the proliferation of either human or rat peripheral blood mononuclear cells, in the human system via the Indoleamine/kynurenine axis and in the murine context via nitric oxide/nitrite. Third, ABCB5+ MSCs decreased TNF-α secretion after lipopolysaccharide stimulation and modulated phagocytosis and in both human and rat macrophages, involving prostaglandin E2 and TGF-β1, respectively. Fourth, clinical-grade ABCB5+ MSCs grafted intravenously and intraperitoneally to a cisplatin-induced AKI murine model exerted modulatory effects on mRNA expression patterns toward an anti-inflammatory and pro-regenerative state despite an apparent lack of amelioration of renal damage at physiologic, metabolic, and histologic levels. Our results demonstrate anti-inflammatory and pro-regenerative effects of clinical grade ABCB5+ MSCs in vitro and in vivo and suggest potential therapeutic utility of this cell population for treatment or prevention of cisplatin chemotherapy-induced tissue toxicity.

Radioprotective effects of linden honey in rat peripheral blood

Radiotherapy affects not only malignant, but also a healthy tissue adjacent to tumor by increasing reactive oxygen species generation, with consequent damage to biomolecules, such as the oxidation of membrane lipids, known as lipid peroxidation. The end product of lipid peroxidation is malondialdehyde. Radioprotectors are compounds that could significantly protect normal cells from radiation, without changing the tumor cell radiosensitivity. Synthetic radioprotectors usually have side effects and are toxic. Natural radioprotectors exert protection without adverse effects. In this study, we examined the radioprotective ability of linden honey in rat blood, by detecting alterations in the activities of antioxidant enzymes catalase and glutathione peroxidase and malondialdehyde concentration after the exposure to a therapeutic dose of gamma rays. Sixteen rats were randomly divided into Control and Honey groups. Honey group received honey (1.5 mL(kgd-1)) orally for four weeks, while at the same time Control group were given distilled water. After four weeks, blood was sampled from all animals. Samples were halved, and one series of samples were gamma irradiated (2 Gy). Radiation induced decreased glutathione peroxidase activity and increased malondialdehyde level, while honey treatment attenuated those alterations, keeping glutathione peroxidase and malondialdehyde at physiological levels. These findings confirm radioprotective properties of linden honey.

Analysis of the influence of enzyme preparations produced using technological microorganisms on the microbiome and cellular immunity of rats

The observed increase in the production of enzyme preparations (EP) using mutant and genetically modified microorganisms makes it necessary to assess their risks to consumer health. However, at present, their possible influence on the microbiome, immune status of the macroorganism has not been sufficiently studied. The purpose of the research was to assess the effect of two EP - the complex of hydrolases with proteolytic and nuclease activity from the Aspergillus oryzae RCAM 01134 mutant strain (EP1) and the neutral protease - bacillolysin and serine protease from the Bacillus subtilis-96 (VKM B-3499D) mutant strain (FP2) on the intestine microbiome and cellular immunity indices of the experimental animals. Material and methods. The experiment on the subacute toxicity of EP1 and EP2 was carried out for 30 days using Wistar rats (7 groups of 10 males each). EP was administered to animals intragastrically in doses 0 (control - saline solution); 1, 10 and 100 mg/kg body weight per day. The composition of the cecum microbiocenosis was studied by inoculating tenfold dilutions of the cecum contents on the differential diagnostic media. The quantitative content of short-chain fatty acids (SCFA - acetic, propionic, isobutyric, butyric, isovaleric, valerianic acids) in the colon contents was determined by highperformance gas chromatography. The expression of T- and B-lymphocyte and NK-cell receptors (CD45RА+, CD3+, CD161+, CD3+CD4+, CD3+CD8+) in rat peripheral blood was determined using an FC-500 flow cytometer. Results. The data obtained as a result of the microbiocenosis studies of the cecum contents indicate that EP1 and EP2 administration had a reliable effect on the quantitative and qualitative composition of aerobic microorganisms, including opportunistic microorganisms, as well as it lead to a weak increase in the number of bifidobacteria and lactobacilli. The development of an inflammatory process in animals of all experimental groups have been caused by the changes in the qualitative and quantitative composition of various groups of microorganisms in the cecum contents, SCFA level in the faeces and indicators of cellular immunity under intragastric administration of FP1 and FP2 for 30 days. Conclusion. The revealed difference in the composition of the cecum microbiocenosis, SCFAs produced by the intestinal microflora, and the cellular immunity indices of the experimental animals under EP1 and EP2 administration, in our opinion, is due to the spectrum of metabolites produced by the intestinal microflora, as well as strains of Aspergillus oryzae RCAM 01134 and Bacillus subtilis-96 (VKM B-3499D). The features of the relationship between the spectrum of SCFAs produced by the intestinal microflora, their quantity and the percentage of T- and B-lymphocytes in the blood of rats indicate different mechanisms of the influence of EP1 and EP2 on the microbiome and immune status of the macroorganism.

The condition of the intracellular metabolism of peripheral blood leukocytes in arterial hypertension (experimental study)

Objective: the comparative analysis of the features of intracellular metabolism of polymorphonuclear leukocytes (PNL) in the peripheral blood of rats with the normal blood pressure and hereditary stress-induced arterial hypertension (ISIAH). Material and methods. Indicators of carbohydrate and lipid metabolism, activity of some key redox enzymes in leukocytes of rats as well as a level of PNL functional activity were examined by histochemical methods. Results. The study determined that the stable increased blood pressure is accompanied by hypertrophy of the left ventricle of the heart, degenerative changes in cardiomyocytes and the decreased microcirculatory bed density. In this case amount of intracellular glycogen in leukocytes was decreased in 23% at unchanged amount of intracellular lipids. Activities of intracellular ATP-ase and myeloperoxidase were reduced in 22% with non-significant increasing of succinate dehydrogenase activity (in 13%). Amount of PNL with positive NBT-test was increased in 35% in the rats of ISIAH. Conclusions. Analysis of the studied metabolic indicators of PNL shows disturbance of the energy-supplying and enzymatic processes in the leukocytes on the background of arterial hypertension.

Diabetes-correcting and antioxidant effects of grape pomace extract rich in natural complex of polyphenols

Background. The positive health effects of polyphenols have led to an increased scientific interest in these natural compounds over the past decade. Many studies confirm the effectiveness of polyphenols as additional therapy in diabetes, especially due to the sugar-lowering effect of polyphenols. The aim of the research was to investigate the morphological and functional state of peripheral blood erythrocytes and the indices of oxidative stress in the liver of rats with experimental diabetes and after the administration of grape pomace extract rich in natural complex of polyphenols. Materials and Methods. We obtained grape pomace extract, which contains a variety of polyphenolic compounds. Rats of the following groups were used in the experiments: control animals, animals treated with grape pomace extract rich in natural complex of polyphenols for 14 days, animals with streptozotocin-induced diabetes mellitus, and animals with streptozotocin-induced diabetes mellitus treated with grape pomace extract rich in natural complex of polyphenols for 14 days. The number of erythrocytes and reticulocytes, the concentration of hemoglobin and glycated hemoglobin were determined in the peripheral blood of rats. The activities of catalase, superoxide dismutase and the content of thiobarbituric acid-reactive substance and carbonyl groups of proteins were determined in the liver tissues of rats. Results. The study has shown an increase in the number of erythrocytes and the level of hemoglobin, a decrease in the level of glycated hemoglobin and the number of reticulocytes in the peripheral blood of rats after administration of grape pomace extract rich in natural complex of polyphenols to rats with experimental diabetes. A decrease in the content of thiobarbituric acid-reactive substance and the content of carbonyl groups of proteins of neutral and basic character and an increase in the activity of catalase and superoxide dismutase in liver tissues were found under the same conditions. Conclusions. The results indicate that the extract of the natural complex of polyphenols is capable of correcting the morphological and functional state of erythrocytes, as well as improving the activity of antioxidant enzymes and the content of marker molecules of oxidative stress in hepatocytes of rats under experimental diabetes mellitus.

Role and mechanism of IL-6/STAT3/Th17 signaling pathway in the intervention of forsythia in acute lung injury of septic rats

Objective: To investigate the mechanism of interleukin-6 (IL-6) /signal transducers and activators of transcriptional 3 (STAT3) /helper T cell 17 (Th17) signaling pathway in lung injury of rats with sepsis intervened by forsythia, with a view to provide experimental basis for the role and mechanism of forsythia in the treatment of sepsis. Methods: In July 2021, 30 healthy Wistar male rats were selected and randomly divided into sham operation group, model group and treatment group, with 10 rats in each group. The rat model of sepsis was established by cecal ligation and puncture. 2 h after recovery, the rats were given traditional Chinese medicine forsythia orally, twice a day at an interval of 12 h. The wet/dry mass ratio (W/D) of lung tissue was detected 24 h after surgery. The morphological changes of lung tissue were detected by HE staining. Flow cytometry was used to detect Th17 population in peripheral blood. The expression levels of IL-6 and interleukin-17A (IL-17A) in serum were determined by enzyme-linked immunosorbent assay. The mRNA expression levels of IL-17A and IL-6 in lung tissues were detected by reverse transcription-polymerase chain reaction. The expression levels of STAT3 and IL-17A in lung tissue were determined by Western blotting. Results: Compared with model group, the W/D of lung tissue in treatment group was decreased, and the difference was statistically significant (P<0.05). HE staining of lung tissue showed that compared with the model group, the degree of lung lesion and injury was reduced in the treatment group. Compared with sham operation group, the proportion of Th17 cells in CD4 lymphocytes in peripheral blood of rats in model group was significantly increased (P<0.05). Compared with model group, the proportion of Th17 cells in CD4 lymphocytes in peripheral blood of rats in treatment group was significantly decreased (P<0.05). Compared with sham operation group, peripheral blood serum IL-6 and IL-17 of rats in model group were significantly increased (P<0.05). Compared with model group, IL-6 and IL-17 in peripheral blood serum of rats in treatment group were decreased (P<0.05). Compared with sham operation group, the expressions of IL-17A and IL-6 mRNA in lung tissue of model group were significantly increased (P<0.05). Compared with model group, the expressions of IL-17A and IL-6 mRNA in lung tissue of rats in treatment group were significantly decreased (P<0.05). Compared with sham operation group, the protein expressions of STAT3 and IL-17A in lung tissue in model group were significantly increased (P<0.05). Compared with model group, the pritein expressions of STAT3 and IL-17A in lung tissue in treatment group were significantly decreased (P<0.05) . Conclusion: Forsythia plays a role in alleviating lung injury by down-regulating the expressions of the signaling pathway IL-6/STAT3/Th17, which providing a new target for the treatment of sepsis induced lung injury.

Proteomics Sequencing Reveals the Role of TGF-β Signaling Pathway in the Peripheral Blood of Offspring Rats Exposed to Fluoride.

The underlying mechanism of fluorosis has not been fully elucidated. The purpose of this study was to explore the mechanism of fluorosis induced by sodium fluoride (NaF) using proteomics. Six offspring rats exposed to fluoride without dental fluorosis were defined as group A, 8 offspring rats without fluoride exposure were defined as control group B, and 6 offspring rats exposed to fluoride with dental fluorosis were defined as group C. Total proteins from the peripheral blood were extracted and then separated using liquid chromatography-tandem mass spectrometry. The identified criteria for differentially expressed proteins were fold change > 1.2 or < 0.83 and P < 0.05. Gene Ontology function annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed using the oeCloud tool. The 177 upregulated and 22 downregulated proteins were identified in the A + C vs. B group. KEGG pathway enrichment analysis revealed that transforming growth factor-β (TGF-β) signaling pathway significantly enriched. PPI network constructed using Cytoscape confirmed RhoA may play a crucial role. The KEGG results of genes associated with fluoride and genes associated with both fluoride and inflammation in the GeneCards database also showed that TGF-β signaling pathway was significantly enriched. The immunofluorescence in HPA database showed that the main expression sites of RhoA are plasma membrane and cytosol, while the main expression site of Fbn1 is the Golgi apparatus. In conclusion, long-term NaF intake may cause inflammatory response in the peripheral blood of rats by upregulating TGF-β signaling pathway, in which RhoA may play a key role.