Supernatants Of Tissue Homogenates Research Articles

Assessment of ∆9-THC and ∆9-THCCOOH Bias, Precision, and Ionization Suppression/Enhancement between Solid Tissue Homogenate and Supernatant by LC-MS/MS.

LC-MS-MS assays are frequently utilized for screening and confirmatory purposes in the forensic toxicology laboratory. While these techniques are excellent for the targeted identification and quantitation of a wide variety of drug classes, validation and determining fit-for-purpose is a requirement for each method. In the United States, ANSI/ASB Standard 036 currently serves as a primary resource in forensic toxicology method validation, and mandates that laboratories evaluate critical performance characteristics to help ensure the production of forensically defensible results. Due to the variability of specimen quality frequently encountered in the discipline of postmortem toxicology, the [Author Information Removed] Office of the Chief Medical Examiner Forensic Toxicology Laboratory routinely analyzes solid tissue specimens as part of the medicolegal death investigation process and evaluates liver as a representative solid tissue matrix during method validation. Authentic postmortem specimens (e.g., liver, kidney, skeletal muscle, and spleen) were used to investigate the effects of analyzing solid tissue homogenate versus solid tissue supernatant on bias, precision, and ionization suppression/enhancement of ∆9-THC and ∆9-THCCOOH. Bias was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant with a single exception of the low QC concentration for Δ9-THC in liver homogenate (-29%). Within-run and between-run CV was <20% for Δ9-THC and ∆9-THCCOOH in liver homogenate and supernatant. Δ9-THC and Δ9-THC-d3 exhibited significant ion suppression in both liver homogenate and supernatant, while ∆9-THCCOOH and ∆9-THCCOOH-d3 showed both ion suppression and enhancement in these matrices. Noticeable quantitative differences were observed in authentic postmortem solid tissue homogenate and supernatant specimens despite evaluating from identical tissue samplings. A brief discussion of the results is presented using a validated LC-MS-MS method for the confirmation and quantitation of ∆9-THC and ∆9-THCCOOH in postmortem casework.

Agarwood extract mitigates alcoholic fatty liver in C57 mice via anti‑oxidation and anti‑inflammation.

Alcoholic fatty liver disease (AFLD) is a disease with a high incidence rate among individuals who drink alcohol. Our previous study found that agarwood alcohol extracts (AAEs) have a protective effect against drug‑induced liver damage via anti‑inflammatory and antioxidant mechanisms. Therefore, we hypothesized that agarwood may have a protective effect against AFLD. The present study assessed the potential protective effects and the underlying mechanism of action of AAEs for the treatment of an AFL invivo model. The AFLD mouse model was established by continuous high fat diet and alcohol gavage in C57 mice. After treatment with AAEs, blood was collected, liver and adipose tissues were removed and liver and adipose indexes were analyzed. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG) and cholesterol (CHO) in serum were detected. The liver tissue was assessed using pathological sections. Biochemical methods were used to detect the levels of oxidative stress in the supernatant of liver tissue homogenate. The levels of pro‑inflammatory cytokines in the serum were detected by ELISA. The protein expression levels of nuclear erythroid 2‑related factor 2 (Nrf2) and nuclear factor kappa‑B (NF‑κB) in liver tissues were detected using western blotting. AAE treatment decreased the liver and adipose indexes, reduced the levels of AST, ALT, TG and CHO, improved the liver pathological characteristics and enhanced antioxidant and anti‑inflammatory activities. In addition, AAEs increased the protein expression level of Nrf2 and decreased the protein expression level of NF‑κB compared with AFL mice. AAE‑treated animals exhibited reduced metabolic enzyme and blood lipid levels, demonstrated improved liver function and relieved the pathological damage of AFLD induced by consuming a high fat and alcohol diet. AAEs have potential protective effects in AFLD via antioxidant and anti‑inflammatory mechanisms.

Prognostic value of the levels of CTLA-4 and its ligand B7.2 in patients with colorectal cancer

Aim. To develop a computer program to determine the probability of colorectal cancer based on the assessment of the levels of CTLA-4 and its ligand B7.2.Materials and methods. The study included 44 patients with colorectal cancer (CRC) and 25 patients with benign tumors of the colon. The control group consisted of 25 individuals who had been operated for colon injury. We determined the levels of CTLA-4 and B7.2 in the blood serum and in the supernatants of tumor tissue and lymph node homogenates using flow cytofluorometry.Results. We found that the level of CTLA-4 in the blood serum increased by 2.77 times in CRC patients compared to the control group (p &lt; 0.001). The concentration of CTLA-4 in the tumor tissue in patients with CRC was 2.34 times higher than in the control group (p = 0.007). The concentration of the B7.2 ligand in the blood serum of patients with CRC exceeded this parameter in the control group by 2.51 times (p = 0.002). The concentration of B7.2 in the tumor tissue of CRC patients was 1.68 times higher (p = 0.004) than in the control group. The analysis of the obtained data determined the parameters that have prognostic value in the structure of the diagnostic model. Using these parameters, we developed a computer program to determine the probability of CRC in the patient.Conclusion. The data obtained demonstrate an increase in the levels of CTLA-4 and its ligand B7.2 in the serum and tumor tissue of patients with CRC.

Differentiation of umbilical cord mesenchymal stem cells into hepatocytes with CYP450 metabolic enzyme activity induced by a liver injury microenvironment

The aim of this study was to observe the effect of a simulated liver tissue injury microenvironment on the directed differentiation of umbilical cord mesenchymal stem cells into hepatocytes with CYP450 metabolic activity in vitro, and to explore the mechanisms underlying this directed differentiation. Normal and damaged liver tissue homogenate supernatants (LHS and CCl4-LHS, respectively) were used as induction fluids. After induction for different durations, Western blot and RT-PCR were used to measure the protein and gene expression of the hepatocellular proteins AFP, CK18, ALB, and the CYP450 family. Simultaneously, the metabolic activity of CYP450 in hepatocytes was determined. Compared with the LHS and CCl4-LHS controls, the LHS and CCl4-LHS induction groups showed a significantly elevated protein and gene expression of AFP, CK18, ALB, CYP1A1/2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 (P < 0.05). The metabolic activity of CYP450 in hepatocytes was increased (P < 0.05). In addition, compared with the LHS group, the CCl4-LHS group induced cell differentiation more rapidly and with a higher efficiency. The results suggested that a liver injury microenvironment is conducive for the directed differentiation of umbilical cord mesenchymal stem cells into hepatocytes with metabolic enzyme activity.

Dahuang Mudan decoction repairs intestinal barrier in chronic colitic mice by regulating the function of ILC3

Ethnopharmacological relevanceDahuang Mudan decoction (DMD) is a classic prescription for treating intestinal carbuncle from Zhang Zhongjing's “Essentials of the Golden Chamber” in the Han Dynasty. Recent studies also prove that DMD has a therapeutic effect on ulcerative colitis (UC), but its mechanism is still unclear. Aim of studyIn this study, we aim to assess the therapeutic effect of DMD on DSS-induced chronic colitis in mice and deeply expound its underlying regulative mechanism. Materials and methodsThe efficacy of DMD on mice with 2% DSS-induced chronic colitis was examined by changes in mouse body weight, DAI score, colon length changes, peripheral blood white blood cells (WBC) and red blood cells (RBC) counts, and hemoglobin (HGB) content, using mesalazine as a positive control. A small animal imaging system observed the FITC-Dextran fluorescence distribution in mice, and the contents of IL-22 and IL-17A in colon tissue homogenate supernatant and LPS in peripheral blood were detected by ELISA. Fluorescence in situ molecular hybridization and bacterial culture were used to investigate bacterial infiltration in intestinal mucosa and bacterial translocation in mesenteric lymph nodes and spleen. Mice immune function was further evaluated by analyzing the changes in spleen index, thymus index, and the ratio of peripheral blood granulocytes, monocytes, and lymphocytes. Meanwhile, the proportion of NCR+ group 3 innate lymphoid cells (ILC3), NCR-ILC3, and IL-22+ILC3 in colonic lamina propria lymphocytes of mice was detected by flow cytometry. The contents of effectors IL-22, IL-17A, and GM-CSF were detected by RT-PCR. We use cell scratching to determine the effect of DMD conditioned medium on the migration of Caco-2 cells by establishing an in vitro model of MNK-3 conditioned medium (CM) intervening Caco-2 cells. RT-PCR and WB detect the expression of tight junction ZO-1, Occludin, and Claudin-1. ResultsDMD restored the body weight, colon length, peripheral blood RBC numbers, and HGB content of chronic colitis mice and reduced peripheral blood WBC and colon inflammatory cell infiltration. Moreover, DMD decreased LPS content in serum, bacterial infiltration of colonic mucosa, and bacterial translocation in spleen and mesenteric lymph nodes. Simultaneously, DMD intensified the expression of ZO-1, Occludin, and Claudin-1, the ratio of NCR+ILC3 and IL-22+ILC3, and decreased the proportion of NCR-ILC3. In vitro studies also confirmed that the conditioned medium of DMD promoted the migration of Caco-2 cells and the expression of tight junction proteins. ConclusionOur results confirm that DMD improves inflammation and restores intestinal epithelial function in mice with chronic colitis, and the mechanism may be related to regulating ILC3 function.

Mechanism of TLR4 mediated immune effect in transfusion-induced acute lung injury based on Slit2/Robo4 signaling pathway

BackgroundTransfusion-related acute lung injury (TRALI) is the infusion of blood or blood system. ObjectiveTo explore the mechanism of TLR4-mediated T cell immune effect in TRALI. MethodsIn this animal study, a mouse model of LPS-induced TRALI was established. Sixty adult C57/BL6 mice (wild-type, WT) were randomly divided into 5 groups: 1) normal WT type, 2) LPS control group of WT type lipopolysaccharide, 3) WT type TRALI group (LPS + MHC-I mAb), 4) (TLR4 antibody) lipopolysaccharide LPS control group, 5) (TLR4 antibody) TRALI group (LPS + MHC-I mAb). Mice were injected with LPS (0.1 mg/kg) and MHC-I mAb (2 mg/kg) into the tail vein. H&E staining was performed to detect pathological features. The myeloperoxidase (MPO) activity and the level of inflammatory cytokines in lung tissue homogenate supernatant were measured. Blood, spleen single-cell suspension, and bronchoalveolar lavage fluid were collected to detect the ratio of Treg and Th17 cells by flow cytometry. RT-PCR and WB were used to detect mRNA or protein expression. ResultsTLR4 mAb treatment alleviated the pathogenesis of LPS-induced TRALI in vivo, the MPO activity, and the level of proinflammatory factors in lung tissues. TLR4 exerted its function by changing of Treg/Th17 ratio via the SLIT2/ROBO4 signaling pathway and downregulating CDH5 and SETSIP. ConclusionTLR4 mediates immune response in the LPS-induced TRALI model through the SLIT2/ROBO4 signaling pathway.

Ameliorating Effect of Bacopa-Monnieri against Nicotine Induced Cerebellar Toxicity in Male Sprague-Dawley Rats

Nicotine present in the tobacco leaves are activated through nicotinic acetylcholine receptors and are involved in neurobehavioral activity. Despite creating awareness, there is an increasing trend in the consumption of tobacco. Few plant products have been documented to protect the pathological consequences of nicotine. The present study is aimed to find the beneficial effects of Bacopa-Monnieri (BM) against nicotine induced physical, psychological, neurobehavioral and biochemical changes in cerebellum. Thirty-two male Sprague-Dawley rats (220-235g) were equally randomized into four groups: Group I: Control; received normal Saline. Group II: received Nicotine (5mg/kg Body-Weight) for 90 days. Group III: received nicotine (5mg/ kg Body-Weight) for initial 90 days followed by Bacopa-Monnieri (100mg/kg Body-Weight) for next 90 days. Group IV: received Bacopa-Monnieri (100 mg/kg Body-Weight) for 90 days. All compounds were administered through oral gavage. Rats were subjected to Open Field Test, Elevated Plus Maze and Beam Walking Test. Following behavioral tests, rats were anesthetized with ketamine (80 ml/kg Body-Weight) and xylocaine (100 ml/kg Body-Weight), brain was dissected and cerebellum was separated. Concentration of Malondialdehyde, Nitric Oxide and activity of Glutathione Peroxidase were measured spectrophotometrically in the supernatant of cerebellum tissue homogenates. Nicotine increases the Malondialdehyde (MDA) and Nitric-Oxide (NO) level in cerebellar tissue compared to control. Nicotine induced increase in Malondialdehyde and Nitric-Oxide level were prevented by Bacopa-Monnieri. The Glutathione Peroxidase (GPx) activity was lower in nicotine treated rats whereas oral supplementation of Bacopa-Monnieri significantly increases the activity of Gluathione-Peroxidase. Bacopa-Monnieri supplementations significantly reverse the Nicotine induced reduction in locomotion activity, exploratory behavior, anxiety, motor impairment and balance. Bacopa-Monnieri confers the protective effects against nicotine induced neurobehavioral alteration and oxidative stress in rats.

Protective Effect of Deer Skin Glue Polysaccharide on Alcoholic Liver Injury

Objective: To explore the protective effect of deer skin gum polysaccharide on acute alcoholic liver injury. Methods: The crude deer skin gum polysaccharide was extracted by extraction method, and purified by column chromatography to obtain pure deer skin gum polysaccharide, and its physical and chemical properties were identified by column chromatography, infrared spectroscopy and physical and chemical reaction experiments. The content of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) in mouse serum and the activity of GSH-Px, SOD and the content of MDA in the supernatant of liver homogenate were measured. The liver tissue sections of mice were observed to explore the protective effect of deer skin gum polysaccharide on acute alcoholic liver injury. Results: The deer skin gum polysaccharide was an acid polysaccharide with the pyranose structure. Its shape was white flocculent, with a molecular weight of 41.2 kDa and the purity of 86.71%. It had no starch, phenols, protein, amino acids and reducing sugar residues.The animal experiment results showed that compared with the model group, the deer skin gum polysaccharide middle and high dose groups could significantly increase the SOD and GSH-Px activities in the serum of mice (P<0.05), and reduce the ALT, AST activities and MDA content (P<0.05). At the same time, it could significantly increase the activity of SOD and GSH-Px in the supernatant of liver tissue homogenate (P<0.05), and significantly reduce the content of MDA (P<0.05). Conclusion: Deer skin gum polysaccharide has obvious protective effect on mice with acute alcoholic liver injury.

Sequential administration of PEG-Span 80 niosome enhances anti-tumor effect of doxorubicin-containing PEG liposome

To improve the anti-tumor effect of polyethylene glycol-modified liposome containing doxorubicin (DOX-PEG liposome), the effect of sequential administration of PEG-Span 80 niosome was investigated for Colon-26 cancer cells (C26)-bearing mice. The concept of the current study is as follows: Since both particulates would be accumulated in the tumor tissue due to the enhanced permeability and retention (EPR) effect, PEG-Span 80 niosome, mainly composed of synthetic surfactant (Span 80), would interact with DOX-PEG liposome and be a trigger to induce the release of DOX from the liposome within the tumor tissue, leading to the improvement of anti-tumor effect of DOX-PEG liposome. To find out an adequate liposome for this strategy, several PEG liposomes with different compositions were examined in terms of drug release enhancement and it was found that PEG-Span80 niosome could significantly enhance the release of calcein and DOX from a PEG liposome composed of 90% hydrogenated soybean phosphatidylcholine (HSPC) and 10% cholesterol. The sequential administration of PEG-Span 80 niosome at 24 or 48 h after dosing of DOX-PEG liposome provided a higher anti-tumor effect than the single dose of DOX-PEG liposome in the C26-bearing mice. Particularly, the 24 h-later dosing of PEG-Span 80 niosome has been found to be more effective than the 48 h-later dosing. It was also confirmed that the coexistence of PEG-Span 80 niosome with DOX-PEG liposome in 50% serum or in 50% supernatant of tumor tissue homogenate significantly increased DOX release from PEG liposome, suggesting that DOX release from DOX-PEG liposome within tumor tissue would be enhanced via the interaction with PEG-Span 80 niosome. This strategy would lead to the safer and more inexpensive chemotherapy, since it could make it possible to provide the better anti-tumor effect by utilizing the lower dose of DOX.

Development of immunochromatographic strip assay for rapid detection of novel goose astrovirus

The novel goose astrovirus (GoAstV) is an emerging pathogenic virus that has resulted in large economic losses to the goose-rearing industry in China since 2016. The novel goose astrovirus cause gout in goslings with a mortality rate of around 50 %. Therefore, an effective diagnostic approach to monitor the spread of GoAstV is necessary. Here, a novel diagnostic immunochromatographic strip (ICS) assay was developed to detect GoAstV. A rapid immunochromatographic assay based on antibody colloidal gold nanoparticles specific to GoAstV was developed for the detection of GoAstV in goose allantoic fluid and supernatant of tissue homogenate. Monoclonal antibodies (Mabs) were prepared using the hybridoma technology, and the polyclonal antibodies (Pabs) were generated by immunizing the rabbits with recombinant ORF2 protein. In addition, the colloidal gold was prepared by reducing gold salt with sodium citrate coupled with Mabs against GoAstV. The optimal concentrations of the coating antibody and the capture antibody were examined as 1.6 mg/mL and 6 μg/mL. The optimal pH of the colloidal gold labeling was pH 8.0. With the visual observation, the lower limit of the ICS was reported to be approximately 1.2 μg/mL. Common diseases of goose were examined to assess the specificity of the ICS, and no cross-reaction was identified. 40 clinical positive samples were simultaneously detected by using the ICS and the PCR with a 92.5% coincidence rate between them. Furthermore, the mentioned samples could be stored at 25 °C and 4 °C for 4 and 6 months, respectively. It was proved that the ICS in this study was highly specific, sensitive, repeatable and more convenient to rapidly detect GoAstV in clinical samples.

Infiltration of inflammatory factors induced penile damage in chronic prostatitis.

The present study aimed to investigate the mechanism of penile damages in experimental autoimmune prostatitis (EAP) rat models to reveal the potential pathological mechanism of the relationship between CP and penile damages. Sprague-Dawley (SD) rats were administered with different concentrations of prostate tissue homogenate supernatant (PTHS) by multipoint subcutaneous injection to establish EAP models. IHC staining was done to assess the expression of inflammatory cytokines in prostate tissues and the corpus cavernosum of penis. Masson and Tunel staining was conducted to observe the fibrosis and apoptosis in the corpus cavernosum. Finally, the functional changes of corpus cavernosum were assessed by WB and IHC staining. The results revealed that EAP rats with different prostatitis severity were successfully established by PTHS. The expression of IL-1β, IL-6 and TNF-α in prostate tissues increased with the concentration of PTHS. The results of Masson and Tunel staining indicated fibrosis and apoptosis gradually aggravated in corpus cavernosum among different subgroups. The function of cavernosum impaired by prostatitis from WB and IHC results and positively with the severity. In conclusion, there existed the infiltration of inflammatory factors and impaired function in the corpus cavernosum of EAP rats' penis and positively correlated with the severity of prostatitis.

Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice

Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice

EVALUATION OF ANTIOXIDANT PROPERTIES OF MEDGERM IN ACUTE SIMULATED HEPATITIS

Disruption in redox processes plays a significant role in the pathogenesis of hepatitis. A balance between peroxidation, on the one hand, and the antioxidant system, on the other, is a prerequisite for maintaining normal cell activity. The modern arsenal of effective hepatoprotective drugs is not so wide therefore, the development of new hepatoprotectors is one of the topical issues in pharmacology. The purpose of the work is study the antioxidant properties of medgerma in acute experimental hepatitis. Material and methods. The experiments were performed on male rats weighing 180-220 g. Acute toxic hepatitis in rats was induced by intraperitoneal administration of D-galactosamine in a dosage of 400 mg / kg (LD50). The experimental animals were divided into 4 groups: group I (n = 10) included intact animals that were injected 0.9% sodium chloride solution (control group) intraperitoneally, II group (n = 40) involved the animals receiving only D-galactosamine; group III (n = 40) included rats, which received were administered Medmerg intraperitoneally in a dose of 0.4 mg / kg 7 days before D-galactosamine administration and 7 days after D-galactosamine administration. Group IV (n = 40) included rats, which received the comparison medicine, Essentiale® N, in at a dosage of 5 mg / kg in the same mode. The assessment of lipid peroxidation indices and the antioxidant system indices were carried out on days 1, 3, and 7 after the administration of hepatotoxin in the blood serum and the liver tissue homogenate supernatant. The condition of lipid peroxidation processes in the animals with galactosamine hepatitis was assessed by the content of lipid peroxidation end products in the blood and liver tissue that react with 2-thiobarbituric acid (TBA reactants). The condition of the main components of the antioxidant system was evaluated by the level of reduced glutathione and by the activity of catalase and superoxide dismutase. Results and discussion. It has been established that medgerm enhances the liver resistance to hepatotoxicant, as evidenced by a decrease in the level of lipid peroxidation products and the consumption of main components of the antioxidant system on the first day of hepatitis. Moreover, in the rats, which received Medgerm according to prevention scheme under acute toxic hepatitis, the course of the disease was milder than in the animals that did not receive this therapy. Conclusions and prospects for further research. Medgerm possesses hepatoprotective properties, which underlay positive effects on the state of lipid peroxidation and the main components of the antioxidant system.

The Proteolytic Fraction From Vasconcellea cundinamarcensis Latex Displays Anti-Inflammatory Effect in A Mouse Model of Acute TNBS-Induced Colitis

The proteolytic fraction (P1G10) from Vasconcellea cundinamarcensis, displays gastric protective and healing activities in different skin lesions in mice and human. In an excisional model, this fraction accelerates resolution of lesions and modulates inflammatory mediators. Based on these data, we assessed its anti-inflammatory activity in murine colitis model, induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) adopted by its physiopathological similarity with human colitis. Twenty four hours after colitis induction followed by three days of treatment, P1G10 at 0.3 and 3.0 mg/Kg induced 30% increase in body weight (p < 0.0001) and ~80% reduction in colon macroscopic damage score (p < 0.05) compared to the untreated TNBS-induced colitis group. Histological analyses showed that 0.3 mg/Kg P1G10 reduced the inflammatory profile and tissue damage (47%, p < 0.05) when it was proteolytically active. Compared to TNBS group, 0.3 mg/Kg P1G10 reduced MPO activity (80%, p < 0.01), MCP-1 (47%, p < 0.05) and TNF-α (50%, no significant) and increased IL-10 (330%, p < 0.001) levels in the supernatant of colonic tissue homogenate. P1G10 treatment also reduced COX-2 expression (60%, p < 0.05) and metalloprotease-2 activity (39%, p < 0.05) while increased globet cell density (140%, p < 0.01), that contributes to mucus layer protection in colonic tissue. Taken together, these findings suggest that low doses of active P1G10 promotes lesion resolution, at least in part by its anti-inflammatory activity, in TNBS-colitis model.

Treatment with a Zinc Metalloprotease Purified from Bothrops moojeni Snake Venom (BmooMP-Alpha-I) Reduces the Inflammation in an Experimental Model of Dextran Sulfate Sodium-Induced Colitis.

It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 μg/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 μg/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 μg of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 μg treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition.

Antidiarrheal effects of a thermostable protein fraction obtained from the larvae of Musca domestica

Our objective was to investigate whether a thermostable protein fraction (TPF) obtained from the larvae of Musca domestica, which contains cecropin family AMPs, is effective in treating senna leaf (Folium Sennae)-induced diarrhea in mice and its possible underlying mechanism. We did the experiments both in vitro and in vivo. Firstly, lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 macrophages. The expression level of nitric oxide (NO) and tumor necrosis factor (TNF)-α was assessed using kits and immunofluorescence assay. A mice model of total diarrhea was established using a decoction of Folium Sennae. Levels of interleukin (IL)-6 and IL-1β in mice serum and of TNF-α in the supernatant of jejunal tissue homogenate were measured using commercially available ELISA kits. Hematoxylin–eosin staining was employed to evaluate pathological lesions, and immunohistochemistry was used for determining IL-1β, IL-6, and TNF-α expression levels. Results display that TPF markedly inhibited NO and TNF-α production in LPS-stimulated RAW 264.7 macrophages in vitro. Moreover, TPF significantly lowered the diarrhea index (DI) in diarrheic mice; when TPF was administered at a high dose (120 mg/kg) to mice, in comparison with mice in the model group, DI was markedly reduced. TPF could also decrease the expression levels of some pro-inflammatory factors, high dose TPF treated mice were with the reduction of (202.29 ± 18.58) pg/ml (tumor necrosis factor alpha, TNF-α), (53.69 ± 7.83) pg/ml (interleukin (IL)-1β, IL-1β), (48.44 ± 3.77) pg/ml (IL-6I, L-6) to the model separately. In comparison with berberine hydrochloride, which was used as the positive control in this study, TPF could confer better intestinal protection. To conclude, our results demonstrate that TPF has potent anti-inflammatory activities in vitro and antidiarrheal effects on mice with Folium Sennae-induced diarrhea.

Effects of inner-heating acupuncture on apoptosis of chondrocytes and expression of Caspase-3 and Caspase-9 in rats with knee osteoarthritis

To investigate the effect of inner-heating acupuncture on apoptosis of chondrocytes and expression of Caspase-3 and Caspase-9 in rats with knee osteoarthritis (KOA). A total of 32 rats were divided into a normal group, a model group, a control treatment group and a treatment group by random number grouping method, 8 rats in each one. The rats in the normal group received no intervention. The rats in the remaining three groups adopted modified Videman method to develop KOA model, the ankle joint of left posterior leg was fully extended and fixed with a resin bandage for 6 weeks. After successful modeling, the rats in the model group received no intervention. The rats in the control treatment group were treated with medium-frequency pulse electrotherapy. The rats in the treatment group were treated with inner- heating acupuncture, 30 min each treatment, once a day, five days per week, and totally 3-week treatment was given. After 3 weeks, the damaged cartilage tissue was collected, and HE staining was used to observe the pathological changes of the cartilage tissue of the knee joint. ELISA was used to detect the content of cytochrome-C in the tissue homogenate supernatant. The chondrocytes in damaged cartilage tissue were isolated, flow cytometer was used to detect the changes of apoptosis and mitochondrial membrane potential. The mRNA and protein expression of Caspase-3 and Caspase-9 in chondrocytes were detected by real-time quantitative PCR (qRT-PCR) and Western blot (WB), respectively. Compared with the normal group, the damage of cartilage tissue in the model group was significant, and the expression level of Cyt-C in the homogenate supernatant of damaged cartilage tissue was increased (P<0.01); the chondrocyte apoptosis was increased significantly (P<0.01); the chondrocyte mitochondrial membrane potential was decreased significantly (P<0.01); the mRNA and protein expression of Caspase-3 and Caspase-9 was increased significantly (all P<0.01). Compared with the model group, the cartilage injury in the control treatment group and the treatment group was significantly relieved; the expression level of Cyt-C in the supernatant of damaged cartilage tissue homogenate was decreased (both P<0.01); the chondrocyte apoptosis was significantly reduced (both P<0.01); the chondrocyte mitochondrial membrane potential was increased significantly (both P<0.01). Moreover, the mRNA and protein expression of Caspase-3 and Caspase-9 was significantly reduced (all P<0.01). Compared with the control treatment group, the treatment group was more effective in the treatment of KOA. The inner-heating acupuncture could significantly improve the pathological changes of KOA rats, inhibit the apoptosis of chondrocytes, which may be closely related to the suppression of Caspase-3 and Caspase-9 expression.

Profiling of Cytokine and Chemokine Responses Using Multiplex Bead Array Technology.

Multiplex bead array technology expands upon the principles of the enzyme-linked immunosorbent assay by allowing the simultaneous quantification of a large number of cytokines and chemokines within a single sample. This allows researchers more freedom and opportunities to investigate complex immune responses both in vivo and in vitro. Here we describe and update the detailed assay protocol and technical tips for simultaneous quantification of multiple cytokines and chemokines in mouse biological fluids such as sera, bronchoalveolar lavage fluid, tissue homogenate supernatant, and tissue culture supernatant, using a multiplex bead array assay.

Isolation &amp; Identification of FAV-4 Tianjin Strain and Pathogenicity to Chicks

The Hydropericardium-hepatitis Syndrome (HHS) caused by Fowl adenovirus 4 (FAV-4) is a new epidemic disease in recent years and firstly reported in Tianjin, 2016. The isolation, identification and pathogenicity of FAV-4 Tianjin strain were studied in the paper for providing the theoretical basis to the law of occurrence, development and termination. The virus was isolated and identified from dead & sick birds on a chicken farm in Tianjin by chicken embryo inoculation with tissue homogenate supernatant, PCR and sequencing experiment with viral allantoic fluid; And the pathogenicity was proceeded by inoculating the isolate into 100 one-day-old chicks, which divided into group A, B, C (experimental group) and group D (control), the symptoms, mortality and morphological changes were observed daily, meanwhile the virus was detected by PCR to be sure the same as inoculating strain. The results show that the isolate was 665bp in electrophoresis, had homology of 98.3%-97.3% with FAV-4 by sequencing analysis and finally named as FAV-4 TJ1607 strain in GenBank; They also show in Pathogenicity research that the symptoms were appeared on the 3nd day, death on the 4th day and the peak of death on the 5th day and end of the 8th day in group A and B; The mortality was 64% in group A and 72% in group B; Hepatic edema & congestion, large amounts of basophilic intranuclear inclusions, hydropericardium, parechymal myocarditis and pulmonary edema were observed obviously on the 4th day in group A and B; ALL above changes were the same in group C just appeared 3 days later except death trend and mortality for 32%; And FAV-4 TJ1607 was detected in all experimental groups. The measures of prevention and treatment will be recommended in poultry industry accordance with the HHS disease model.

ADDITIVE EFFECT OF GENES POLYMORPHISM OF FOLATE CYCLE PROTEINS AND HOMOCYSTEIN LEVEL IN PATIENTS WITH PROLIFERATIVE DISEASES OF THE BREAST AS A POTENTIAL FACTOR OF THE RISK OF THROMBOSSES

Aim.The aim of study was to examine the relationship between serum and mammary gland homocysteine levels with the carrier of separate SNP (single nucleotide polymorphism) genes of the folate metabolism system in patients with proliferative diseases and breast cancer.Methods and results.The study included 182 patients with proliferative diseases of the mammary gland in transbaikalia. The control group included 144 women who did not have oncological diseases. The serum homocysteine level and the supernatant of the mammary tissue homogenate were evaluated by high performance liquid chromatography. Genotyping for the detection of polymorphism MTHFRС677T, MTHFRА1298C, MTRA2756G, MTRRA66G was carried out by polymerase chain reaction with the detection of the amplification product in real time. In the course of molecular genetic testing in patients with proliferative diseases of the mammary gland, there was found: 1) the absence of an explicit association of the carriage of genetic polymorphism MTHFRС677T, MTHFRА1298C, MTRA2756G and MTRRA66G with serum homocysteine concentration, however, comparative hyperhomocysteinemia and, to a lesser extent, in women with the benign breast diseases; 2) the highest homocysteine content in the blood in patients with breast cancer whose genotype was characterized by combinations of polymorphic alleles MTR2756G x MTRR66G; 3) that the MTR2756A allele and genotype MTHFR1298AC, especially their combination of MTHFR1298AC x MTR2756A, increase the risk of developing benign breast formations; 4) the effect of the risk alleles MTR2756G and MTRR66GON the concentration of homocystein in the tumor tissue of the mammary gland.Conclusion. These patterns indicate a certain contribution of the polymorphisms studied, especially their additive effect, both in the development of proliferative diseases of the mammary gland and in the possible potentiation of prothrombotic effects in these patients against the background of tumor progression and homocysteine metabolism disorders.