Enzymes In Tissues Research Articles

Dual targeting of H2S synthesizing enzymes; cystathionine β-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer

IntroductionHydrogen sulfide (H2S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H2S synthesizing enzymes; Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. ObjectivesFirstly, this study aimed to demonstrate a comparative expression profile of H2S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H2S synthesizing enzymes that could cease H2S production and reduce TNBC pathogenicity. MethodsPairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H2S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H2S levels were detected using a AzMc fluorescent probe. ResultsBC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H2S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H2S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. ConclusionThis study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H2S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.

Intracellular Gemcitabine Monophosphate Levels Predict Chemotherapy Efficacy in Gemcitabine-Treated Patients with Bladder Cancer.

Gemcitabine monophosphate (dFdCMP), one of the intracellular forms of phosphorylated gemcitabine, determines its antitumor activity. A pharmaco-molecular model for determining relative gemcitabine monophosphate level based on the assessment of the activity of ENT1 and ENT2 channels as well as dCK and CDA enzymes in tumor tissue was developed. Relative gemcitabine monophosphate level is a more relevant predictive factor of gemcitabine resistance of bladder cancer as compared with the expression of individual markers related to dFdCMP formation.

Drug-Induced Acute Pancreatitis in Adults: Focus on Antimicrobial and Antiviral Drugs, a Narrative Review.

Acute pancreatitis (AP) is an acute inflammation of the pancreas caused by the activation of digestive enzymes in the pancreatic tissue. The main causes of AP are cholelithiasis and alcohol abuse; less commonly, it can be caused by drugs, with a prevalence of up to 5%. Causal associations between drugs and pancreatitis are largely based on case reports or case series with limited evidence. We reviewed the available data on drug-induced AP, focusing on antimicrobial drugs and antivirals, and discussed the current evidence in relation to the classification systems available in the literature. We found 51 suspected associations between antimicrobial and antiviral drugs and AP. The drugs with the most evidence of correlation are didanosine, protease inhibitors, and metronidazole. In addition, other drugs have been described in case reports demonstrating positive rechallenge. However, there are major differences between the various classifications available, where the same drug being assigned to different probability classes. It is likely that the presence in multiple case reports of an association between acute pancreatitis and a drug should serve as a basis for conducting prospective randomized controlled trials to improve the quality of the evidence.

Effect of White Noise and Light Exposure on Some Enzymes in Rat Testicular Tissue

By definition, white noise (WN) is a type of sound that consists of a combination of various frequencies. Noise is an important source of environmental stress for humans and other living things and has a strong stimulating effect on light, circadian rhythm, hormonal, and neuro-behavioral levels. When faced with stressful situations, organisms produce a substantial quantity of free radicals, leading to alterations in the oxidative system. The objective of this research is to examine how environmental stressors like WN and light impact the activities of enzymes involved in testicular carbonic anhydrase (CA), catalase (CAT), and glutathione peroxidase (GPx). A total of 18 male Sprague-Dawley rats were divided into three groups. The first was the control group (not exposed to stress, kept at 12/12 light-dark cycle and 24±2°C normal ambient temperature). The second group, known as the WN 60' group, experienced 60 minutes of white noise exposure per day for 14 consecutive days at 90 dB. The third group, referred to as the WN 60' + non daylight group, underwent 60 minutes of white noise exposure at 90 dB combined with 24 hours of continuous light exposure every other day. At the conclusion of the study, the testicular tissues were homogenized and centrifuged, followed by the spectrophotometric measurement of enzyme activities.
 As a result, when we compared the control group with both WN and WN + light exposure; A decrease was observed for CA enzyme in both applications, and an increase was observed in CAT and GPx enzymes in both applications.
 As a result, when we compared the control group with both BG and BG + light exposure; A decrease was observed for CA enzyme in both applications, and an increase was observed in CAT and GPx enzymes in both applications.

Potential of banana based cellulose materials for advanced applications: A review on properties and technical challenges

Biocompatibility, biodegradability, and toxicity issues of synthetic polymers have propelled the search for environmentally friendly and non-toxic alternatives. In this context, biobased materials have gained much popularity due to their non-toxic, biodegradable, and sustainable nature. Bananas are considered as one of such natural material which fulfil the requirements to be tailored as a biocompatible biopolymer. Banana derived wastes can be used for extraction of commercially important biopolymers like starch, cellulose, nanocellulose and their subsequent utilization in wide variety of applications. Banana derived biopolymers and their bio composites and widely used for medical applications such as wound healing, fabrication of bone plates, cellulose based gate dielectrics, and capacitors for insulin pumps, and pacemakers. In addition, banana based nanocellulose can be used in tissue engineering, biosensing, drug delivery, bioimaging, wound healing, enzyme immobilization and preparation of tablets for oral administration. Moreover, banana-based polymers can be employed in applications such as food packaging, biofuel production, and production of multilayered papers. Considering the potential applications of banana-based nanomaterials, this review work is framed to understand the process of extraction of starch, cellulose, nanocellulose and biopolymers from banana derived wastes with specific emphasis on their extraction methods and composite preparation methods. In addition, it discusses in detail the promising and potential applications of the derived materials in health and environmental sectors. The presented review is a comprehensive discussion on banana-based waste conversion strategies to produce value added products useful in medical and environmental applications.

METTL3 Regulates the m6A Modification of NEK7 to Inhibit the Formation of Osteoarthritis.

Osteoarthritis (OA) is a common degenerative joint disease. The occurrence of OA slowly destroys the soft tissue structure of the patient's joint. Severe cases could lead to disability. Current studies had shown that inhibition of chondrocytes pyroptosis could slow down the progression of OA. Our work aimed to explore the specific mechanisms and ways of regulating this process. In this work, the level of N6-methyladenosine (m6A) in clinical tissues was detected by ribonucleic acid (RNA) m6A dot blot. qRT-PCR (quantitative real-time polymerase chain reaction) was used to detect the messenger RNA (mRNA) expression level of m6A modified enzyme in clinical tissues. MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromid) and flow cytometry were used to detect the effect of sh-METTL3 (methyltransferase like 3) and NIMA-related kinase 7 (NEK7) transfection on chondrocytes pyroptosis in OA. Western blot was used to detect the protein expression levels of pyroptosis-related proteins. ELISA (enzyme-linked immunosorbent assay) was used to measure the protein concentration of inflammatory cytokines. The SRAMP online database was used to predict the m6A site of NEK7. HE staining was used to assess the progression of OA in mice. The level of m6A in clinical samples of OA patients was higher, and METTL3 was significantly higher expressed in clinical samples of OA patients. We provided evidence that low expression of METTL3 inhibited chondrocytes pyroptosis. In addition, Rescue experiments and in vivo experiments had shown that METTL3 in combination with NEK7 inhibited the progression of OA by promoting chondrocytes pyroptosis. METTL3 regulates m6A modification of NEK7 and inhibits OA progression.

Intra-Articular AAV9 α-l-Iduronidase Gene Replacement in the Canine Model of Mucopolysaccharidosis Type I

Mucopolysaccharidosis type I (MPS I), an inherited lysosomal storage disorder characterized by deficiency of α-l-iduronidase (IDUA) activity, causes multisystemic pathology due to sequelae of accumulated heparan and dermatan sulfates (HS and DS), the substrates of IDUA. Current treatments, though life-prolonging, inadequately address skeletal dysplasia and do not forestall progressive and painful degenerative joint disease. Previous studies demonstrated that intra-articular enzyme replacement cleared cellular lysosomal storage and reduced joint inflammation. Three nontolerized MPS I canines were studied to assess safety, efficacy, and durability of IDUA gene replacement therapy delivered via intra-articular injection. After baseline joint tissue biopsies, the right shoulder and stifle of each animal were injected in the intra-articular space with AAV9-IDUA and contralateral joints with AAV9-eGFP. Animals received either 5E11 or 5E12 vector genomes/joint. Necropsy was performed at 2- or 52-week postinjection. All animals tolerated injections without adverse effects. At two weeks, supraphysiologic IDUA enzyme activity was measured in AAV9-IDUA-treated but not AAV9-eGFP-treated synovium, with corresponding normalization of HS content and synoviocyte morphology. The AAV9-IDUA-treated cartilage had normal physiologic levels of IDUA enzyme, reduced but not normalized HS and DS levels compared to untreated MPS I cartilage, and healthy chondrocyte morphology. Liver IDUA transgene and IDUA enzyme activity were identified, as was serum IDUA activity which was 40% of wild-type serum enzyme activity. At 52-week postinjection, AAV9-IDUA-treated synovium and cartilage IDUA enzyme activity declined in both animals, corresponding to high tissue HS and DS levels and severe lysosomal storage. Liver and serum IDUA activity levels were undetectable. A dose-dependent serum anti-IDUA antibody response was observed which, together with loss of transgene with age, likely contributed to decline in tissue enzyme activity and treatment efficacy. Our study demonstrates successful proof-of-concept for intra-articular gene replacement therapy as a treatment for MPS-related joint dysplasia. Our observations suggest the possibility of multimodal gene replacement therapy to address multiple refractory manifestations of MPS I. Subsequent studies, in conjunction with immune tolerization and functional assessments of joint pathology, will investigate this possibility.

Serum Free Fatty Acid Changes Caused by High Expression of Stearoyl-CoA Desaturase 1 in Tumor Tissues Are Early Diagnostic Markers for Ovarian Cancer.

Measurement of serum FFA levels by changes in the expression of fatty acid metabolizing enzymes in tumor tissue would allow early detection of ovarian cancer. In particular, the SCD1-associated FFAs, oleic and arachidic acid, would be powerful new screening tools for early-stage ovarian cancer.

Exploring cardiac impact of oral nicotine exposure in a transplantable Neoplasm Mice Model: Insights from biochemical analysis, morphometry, and molecular docking: Chlorella vulgaris green algae support

Nicotine-induced cardiac tissue damage is a concern for cancer patients, but the exact pathogenesis from nicotine oral exposure is unclear. This study was designed to investigate the impact of nicotine and Chlorella vulgaris (Ch. V) on cardiac glutathione homeostasis, inflammatory response, cardiac damage markers, apoptotic proteins and histopathological findings in an experimentally transplantable neoplasm mouse model (Ehrlich ascites carcinoma; EAC). In the in-vivo experiment, the female Swiss mice were divided into four groups: control, Ch.V (100 mg/kg), Nicotine (100 µg/ml/kg), and a combination group ( Nocotine+ Ch.V) for 40 days. Furthermore, in this study,the effects of C. vulgaris components on caspase-3, TNF-α, and IL-1β activity were explored using Molecular Operating Environment (MOE) docking software to ensure its ability to counteract the toxic effects of nicotine. The results indicated that nicotine has induced significant (P < 0.001) cardiopathic alterations in EAC-bearing mice with changes in cardiac tissue enzymes. C. Vulgaris attenuated the nicotine-induced cardiac glutathione inhibition, suppressed the inflammatory response, exerted antiapoptotic effects, mitigated myocardial injury biomarkers, and repaired cellular and tissue damage. Moreover, the molecular docking results revealed the ability of C. vulgaris to bind with interleukin-1 receptor type 1 (IL1R1) and tumor necrosis factor receptor superfamily member 1 A (TNFRSF1A) in the mice tissues, ameliorating apoptosis and inflammatory processes associated with nicotine-induced cardiotoxicity. This study provides a model for understanding nicotine-induced myocardial injury during experimentally transplantable neoplasm. It highlights C. vulgaris as a beneficial food supplement for cancer patients exposed to nicotine orally.

EXPRESSION OF DE NOVO CERAMIDE SYNTHESIS IN LOCAL HEART FAT DEPO IN PATIENTS WITH CARDIOVASCULAR DISEASES

Introduction. Ceramides are bioactive lipids that are not only structural components of cell membranes, but also second messengers in the transmission of cell signals. Ceramides are associated with risk factors for cardiovascular disease, may be independent predictors of cardiovascular events and proatherogenic mediators. However, due attention has not been paid to the problem of ceramide accumulation in the adipose tissue (AT) of the heart, while the accumulation of ceramides in adipocytes of epicardial and perivascular AT in coronary heart disease (CHD) is of particular interest, since they are localized in close proximity to the coronary vessels and the focus of the lesion. Objective: to evaluate the expression of de novo ceramide biosynthesis enzymes in the adipose tissue of the heart of various locations in patients with coronary heart disease and acquired heart defects. Materials and methods. The study included 30 patients with CAD, the comparison group consisted of 30 patients with acquired valvular disease (aortic stenosis/insufficiency) without CAD. During the operation, biopsies of subcutaneous, epicardial, perivascular adipose tissue (VT) (SAT, EAT, PVAT, respectively) were obtained. Expression of genes encoding de novo ceramide synthesis enzymes (subunits of C1 and C2 serine palmitoyltransferases: SPTLC1, SPTLC2; ceramide synthase 1-6: CERS1-6; dihydroceramide desaturase: DEGS1) was assessed by real-time quantitative polymerase chain reaction (qPCR) with primers synthesized by Evrogen (Moscow, Russia) on the ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, California, USA). Statistical analysis of the results was performed using GraphPad Prism 8 (GraphPad Software). Results. Patients with CAD were characterized by higher levels of SPTLC1 mRNA in subcutaneous and epicardial VT and SPTLC2 mRNA in EAT. DEGS1 expression was maximal in SVT and EAT. The expression of ceramide synthase genes in VT of patients with coronary artery disease had tissue-specific features. Expression of the CERS1 gene, encoding the enzyme ceramide synthase-1, which produces ceramides with a fatty acid residue in its composition of 18 carbon atoms (C18), expression of CERS5 and CERS6, encoding ceramide synthase-5 and -6, synthesizing ceramides C14-C16 was maximum in epicardial adipocytes. At the same time, CERS5 expression was higher than CERS6 expression. Gene expression of CERS2, encoding the enzyme ceramide synthase-2, which produces long-chain ceramides and attaches C20–C24 fatty acyl-CoA to the sphingoid base in ceramide, was maximal in subcutaneous adipocytes. While perivascular VT was characterized by a higher expression of the CERS4 and CERS3 genes, which synthesize very long chain ceramides C26 and higher. In the group of patients with valvular disease, VT samples did not differ in terms of SPTLC1, SPTLC2, CERS1, CERS2, CERS5, and CERS6 mRNA levels. However, high expression of CERS4 and DEGS1 was observed in EAT, while CERS3 and CERS4 were expressed in PVAT. When comparing the level of expression of genes for ceramide synthesis enzymes between the two studied groups, it was found that in patients with IHD, the mRNA level of SPTLC1 in SAT and EAT, SPTLC2 in EAT, CERS2 in all types of AT, CERS4 and CERS5 in EAT, DEGS1 in SAT and EAT was higher in compared with patients with valvular heart disease. Conclusion. Regional fat depots of the heart differed in the level of expression of de novo ceramide biosynthesis enzymes. The results obtained indicate the activation of ceramide synthesis along this pathway in adipocytes of predominantly epicardial localization in coronarogenic pathology, which may contribute to the accumulation of long-chain ceramides in the VT of this localization.

Enzymatic tissue processing after testicular biopsy in non-obstructive azoospermia enhances sperm retrieval.

What is the added value of enzymatic processing of testicular biopsies on testicular sperm retrieval (SR) rates for patients with non-obstructive azoospermia (NOA)? In addition to mechanical mincing, enzymatic digestion increased SR rates in testicular biopsies of NOA patients. Many studies focus on the surgical approach to optimize recovery of testicular sperm in NOA, and in spite of that, controversy still exists about whether the type of surgery makes any difference as long as multiple biopsies are taken. Few studies, however, focus on the role of the IVF laboratory and the benefit of additional lab procedures, e.g. enzymatic digestion, in order to optimize SR rates. This retrospective single-center cohort study included all patients who underwent their first testicular sperm extraction (TESE) by open multiple-biopsy method between January 2004 and July 2022. Only patients with a normal karyotype, absence of Y-q deletions and a diagnosis of NOA based on histology were included. The primary outcome was SR rate after mincing and/or enzymes. The secondary outcome was cumulative live birth (CLB) after ICSI with fresh TESE and subsequent ICSI cycles with frozen TESE. Multiple biopsies were obtained from the testis, unilaterally or bilaterally, on the day of oocyte retrieval. Upon mechanical mincing, biopsies were investigated for 30 min; when no or insufficient numbers of spermatozoa were observed, enzymatic treatment was performed using Collagenase type IV. Multivariable regression analysis was performed to predict CLB per TESE by adjusting for the following confounding factors: male FSH level, female age, and requirement of enzymatic digestion to find sperm. We included 118 patients, of whom 72 (61.0%) had successful SR eventually. Spermatozoa were retrieved after mechanical mincing for 28 patients (23.7%; 28/118) or after additional enzymatic digestion for another 44 patients (37.2%; 44/118). Thus, of the 90 patients requiring enzymatic digestion, sperm were retrieved for 44 (48.9%). Male characteristics were not different between patients with SR after mincing or enzymatic digestion, in regard to mean age (34.5 vs 34.5 years), testis volume (10.2 vs 10.6 ml), FSH (17.8 vs 16.9 IU/l), cryptorchidism (21.4 vs 34.1%), varicocele (3.6 vs 4.6%), or histological diagnosis (Sertoli-cell only 53.6 vs 47.7%, maturation arrest 21.4 vs 38.6%, sclerosis/atrophy 25.0 vs 13.6%).Of the 72 patients with sperm available for ICSI, 23/72 (31.9%) achieved a live birth (LB) after the injection with fresh testicular sperm (and fresh or frozen embryo transfers). Of the remaining 49 patients without LB, 34 (69.4%) had supernumerary testicular sperm frozen. Of these 34 patients, 19 (55.9%) continued ICSI with frozen testicular sperm, and 9/19 (47.4%) had achieved an LB after ICSI with frozen testicular sperm. Thus, the total CLB was 32/118 (27.1%) per TESE or 32/72 (44.4%) per TESE with sperm retrieved.Of the female characteristics (couples with sperm available), only female age (30.3 vs 32.7 years; P = 0.042) was significantly lower in the group with a LB, compared to those without.The CLB with testicular sperm obtained after enzymatic digestion was 31.8% (14/44), while the CLB with sperm obtained after mincing alone was 64.3% (18/28). Multivariable logistic regression analysis showed that when enzymatic digestion was required, it was associated with a significant decrease in CLB per TESE (OR: 0.23 (0.08-0.7); P = 0.01). Limitations of the study are related to the retrospective design. However, the selection of only patients with NOA, and specific characteristics (normal karyotype and absence Y-q deletion) and having their first TESE, strengthens our findings. Enzymatic processing increases the SR rate from testicular biopsies of NOA patients compared to mechanical mincing only, demonstrating the importance of an appropriate laboratory protocol. However, NOA patients should be counseled that when sperm have been found after enzymatic digestion, their chances to father a genetically own child may be lower compared to those not requiring enzymatic digestion. None reported. N/A.

Time-dependent changes in oxidative stress biomarkers and activities of lysosomal and antioxidant enzymes in hepatic tissue of rainbow trout (Oncorhynchus mykiss Walbaum) following vaccination against Yersinia ruckeri

Abstract This study analyzed time-dependent effects of vaccination against Y. ruckeri on the oxidative mechanism underlying those effects by detecting relevant lipid peroxidation (2-thiobarbituric acid reactive substances, TBARS) and protein oxidation biomarkers [aldehydic and ketonic derivatives of oxidatively modified proteins (OMP)], antioxidant defenses [activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), total antioxidant capacity (TAC)], as well as activities of lysosomal functioning [alanyl aminopeptidase (AAP), leucyl aminopeptidase (LAP), acid phosphatase (AcP), and β-N-acetylglucosaminidase (NAG)] in hepatic tissue of rainbow trout, Oncorhynchus mykiss (Walbaum) following anti-Y. ruckeri vaccination in the first, second, and sixth months. A concentrated vaccine with Y. ruckeri strains was enclosed in fish feed and was administered three times every other day. Rainbow trout from each group were euthanized 31, 61, and 181 days following vaccination, and hepatic tissue was sampled for analysis. In the current study, vaccination against Y. ruckeri resulted in a no statistically significant change in TBARS levels, while aldehydic and ketonic derivatives of OMP in hepatic tissue decreased, especially after the first and second months following immunization. Moreover, the activities of glutathione-dependent enzymes increased, especially after the first and sixth months. The highest TAC levels were observed two and six months after vaccination. It has been shown that vaccination-related oxidative stress in hepatic tissue is involved in adaptive responses through the temporary mobilization of antioxidant and lysosomal enzymes in rainbow trout. The present study showed the effect of vaccination on lysosome membrane permeability for carbohydrate cleavage after the development of immunity against Yersinia, whereas antioxidant defence was reduced. Our results confirmed that the concept of preserving antioxidant enzyme function after vaccination was also evident when CAT, GR, and GPx activities either increased or were unchanged following vaccination.

Maslinic acid alleviates LPS-induced mice mastitis by inhibiting inflammatory response, maintaining the integrity of the blood-milk barrier and regulating intestinal flora.

Mastitis occurs frequently in breastfeeding women and not only affects the women's health but also hinders breastfeeding. Maslinic acid is a type of pentacyclic triterpenoid widely found in olives that has good anti-inflammatory activity. This study aims to discuss the protective function of maslinic acid against mastitis and its underlying mechanism. For this, mice models of mastitis were established using lipopolysaccharide (LPS). The results revealed that maslinic acid reduced the pathological lesions in the mammary gland. In addition, it reduced the generation of pro-inflammatory factors and enzymes (IL-6, IL-1β, TNF-α, iNOS, and COX2) in both mice mammary tissue and mammary epithelial cells. The high-throughput 16S rDNA sequencing of intestinal flora showed that in mice with mastitis, maslinic acid treatment altered β-diversity and regulated microbial structure by increasing the abundance of probiotics such as Enterobacteriaceae and downregulating harmful bacteria such as Streptococcaceae. In addition, maslinic acid protected the blood-milk barrier by maintaining tight-junction protein expression. Furthermore, maslinic acid downregulated mammary inflammation by inhibiting the activation of NLRP3 inflammasome, AKT/NF-κB, and MAPK signaling pathways. Thus, in a mice model of LPS-induced mastitis, maslinic acid can inhibit the inflammatory response, protect the blood-milk barrier, and regulate the constitution of intestinal flora.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

Studying the possibility of using olive oil and its active components together with atorvastatin in the treatment of biochemical parameters and iron deposition in heart tissue with lung histopathology conditions induced by hypercholesterolemia in male rod

Background Hyperlipidemia is one of the leading causes of the inflammatory response due to damage to the lung tissue, and the antioxidant activity of olive oil and hydroxytyrosol curbs these disorders. Therefore, the current study aimed to investigate the effect of Hydroxytyrosol (HXT) and Olive Oil (OO) on increased levels of Adiponectin, decreased Tumor Necrosis Factor Alpha (TNF-α) and Interleukin 6 (IL-6) in blood serum with the improvement of histological lung disorders in experimental hyperlipidemia in male Sprague Dawley rats and an assessment of outcomes with atorvastatin. Method Thirty albino male rats were used in this study, divided into six groups of approximately identical weight. The initial group received a typical diet devoid of cholesterol. The two groups were fed a diet including 2% cholesterol for the length of the experiment for eight weeks, and the 3rd, 4th, 5th, and 6th teams were placed on a two-week high-cholesterol diet. They were given a dose of (OO) extract just, (HXT) just, (OO) extract + (HXT), and a drug. Atorvastatin (ATOR), consecutively, from the third week to the eighth week of the experiment. Results The results of the study showed a significant decrease (P≤0.05) in the concentration of Adiponectin and a significant increase in the concentration of TNF-α and IL-6 in the blood serum, In addition to a rise in the scale of malondialdehyde (MDA), iron deposition, lower rates in the level of glutathione (GSH), and the activity of the catalase (CAT) enzyme in heart tissue; additionally, numerous lung histopathological disorders, that also comprised damage to the alveolar (DA) and damage to the alveolar sacs (DAS), Thickening Vessels (TV), Thickening Bronchioles (TB), Fibrosis (Fi), Inflammatory Infiltration (II), Congestions (CON), Hemorrhage (H), Degeneration (D), necrosis (N) and karyolysis (KL) in the group with hyperlipidemia food contrasted to the group of healthy controls. Whereas the teams of high-fat rats, which were also gavage by (OO) extract just, (HXT) just, (OO) extract + (HXT), and (ATOR) drug demonstrated an enhancement in each of the factors above, as (OO) extract + (HXT) outperformed all treatments. Conclusions As a result, we conclude the present study that hyperlipidemia causes a significant lowering in the concentration of adiponectin and a significant rise in the concentration of TNF-α and IL-6 in serum from the blood, In addition to lower rates in the scale of MDA, iron deposition, lower rates in the level of GSH, and the activity of the CAT enzyme in heart tissue, as well as many histological disorders in the lung, and that mixing olive oil extract and hydroxytyrosol plays an essential role in Inhibition of adverse consequences of hypercholesterolemia.

Gemcitabine enhances pharmacokinetic exposure of the major components of Danggui Buxue Decoction in rat via the promotion of intestinal permeability and down-regulation of CYP3A for combination treatment of non-small cell lung cancer

Context Danggui Buxue Decoction (DBD), a traditional Chinese medicine formula, has the potential to enhance the antitumor effect of gemcitabine in non-small cell lung cancer (NSCLC) treatment by increasing gemcitabine’s active metabolites. However, whether gemcitabine affects the pharmacokinetics of DBD’s major components remains unclear. Objective This study evaluates the herb–drug interaction between DBD’s major components and gemcitabine and validates the underlying pharmacokinetic mechanism. Materials and methods The pharmacokinetics of 3.6 g/kg DBD with and without a single-dose administration of 50 mg/kg gemcitabine was investigated in Sprague-Dawley rats. The effects of gemcitabine on intestinal permeability, hepatic microsomal enzymes in rat tissues, and CYP3A overexpressing HepG2 cells were determined using western blot analysis. Results The combination of gemcitabine significantly altered the pharmacokinetic profiles of DBD’s major components in rats. The C max and AUC of calycosin-7-O-β-d-glucoside notably increased through sodium-glucose transporter 1 (SGLT-1) expression promotion. The AUC of ligustilide and ferulic acid was also significantly elevated with the elimination half-life (t 1/2) prolonged by 2.4-fold and 7.8-fold, respectively, by down-regulating hepatic CYP3A, tight junction proteins zonula occludens-1 (ZO-1) and occludin expression. Discussion and conclusions Gemcitabine could modulate the pharmacokinetics of DBD’s major components by increasing intestinal permeability, enhancing transporter expression, and down-regulating CYP3A. These findings provide critical information for clinical research on DBD as an adjuvant for NSCLC with gemcitabine and help make potential dosage adjustments more scientifically and rationally.

Identifying hepatoprotective mechanism and effective components of Yinchenzhufu decoction in chronic cholestatic liver injury using a comprehensive strategy based on metabolomics, molecular biology, pharmacokinetics, and cytology

Ethnopharmacological relevanceIn Traditional Chinese Medicine (TCM), cholestasis liver disease belongs to jaundice. Yinchenzhufu decoction (YCZFD) is a classic formula used for treating jaundice. Aim of the studyThis study was aimed to investigate the potential mechanism and effective components of YCZFD in chronic cholestatic liver injury (CCLI). Materials and methodsA chronic cholestatic mouse model induced by 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine was used to investigate the effect of YCZFD. Then, metabolomics was used to investigate the metabolites influenced by YCZFD. Serum and liver bile acid (BA) levels were measured using liquid chromatography coupled with triple quadruple mass spectrometry (LC-MS/MS), and the gene and protein expressions of BA transporters and metabolic enzymes were detected. Additionally, the pharmacokinetics of multiple components of YCZFD was explored to clarify the potential effective components. The effects of absorbed components of YCZFD on BA metabolism and transporter function, inflammation, and farnesoid X receptor (FXR) and pregnane X receptor (PXR) activation were analyzed using sandwich cultured rat hepatocytes, AML12 cells, and dual-luciferase receptor systems, respectively. ResultsYCZFD decreased the liver damage in chronic cholestatic mice. Serum metabolomics results indicated that the main pathways influenced by YCZFD involved primary BA biosynthesis and arachidonic acid metabolism. YCZFD upregulated the expression of FXR, PXR, and BA efflux transporters and the metabolic enzymes of liver tissues, promoting BA excretion and metabolism in cholestatic mice. Additionally, YCZFD downregulated the expression of genes and proteins of the toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway and decreased liver inflammation. The pharmacokinetic study indicated that multiple components showed different pharmacokinetic properties. Among the absorbed components of YCZFD, multiple components activated the transcription of FXR and PXR, regulated BA transporters and metabolic enzyme function, and reduced the gene expression of TLR4 and NF-κB1. ConclusionYCZFD can ameliorate CCLI by promoting the excretion and metabolism of BAs and inhibiting inflammation via the TLR4/NF-κB signaling pathway. The multiple components of YCZFD could act on BA homeostasis regulation and anti-inflammation, exhibiting a combined effect against CCLI.

Gene expression of de novo ceramide synthesis enzymes in the adipose tissue of the heart and lipid spectrum parameters in patients with cardiovascular diseases

Gene expression of de novo ceramide synthesis enzymes in the adipose tissue of the heart and lipid spectrum parameters in patients with cardiovascular diseases

Relationship of the degree of coronary lesion and expression of de novo ceramide synthesis enzymes in the cardiac adipose tissue in coronary artery disease

Relationship of the degree of coronary lesion and expression of de novo ceramide synthesis enzymes in the cardiac adipose tissue in coronary artery disease

Expression of genes for sphingomyelinase and sphingomyelin synthase enzymes in the adipose tissue of the heart of patients with cardiovascular diseases

Expression of genes for sphingomyelinase and sphingomyelin synthase enzymes in the adipose tissue of the heart of patients with cardiovascular diseases