Protein Expression Levels Of TNF-α Research Articles

Involvement of necroptosıs and apoptosıs ın protectıve effects of cyclosporın a on ischemıa-reperfusıon injury in rat kıdney.

We aimed to investigate the protective effects of low dose cyclosporin A (CsA) on ischemia-reperfusion (IR) injury in rat the kidney and on the apoptotic and necroptotic mechanisms involved. 1. Control group (received a single intraperitoneal (i.p.) dose of 1ml sterile saline 15min before the surgical procedure), 2. IR group (was subjected to 30min of bilateral kidney ischemia followed by 90min of reperfusion; and received a single i.p. dose of 1ml sterile saline 15min before the IR procedure, 3. IR + CsA group (received a single i.p. dose of 3mg/kg CsA 15min before the IR procedure. Renal functions (renal perfusion pressures, and serum urea-creatinine levels), kidney histological scores, MDA levels, and TNF-α, caspase-3, RIP1, RIP3, MLKL, CaMKII and CypD protein expressions were also measured. Renal perfusion pressures (PP), serum urea and creatinine levels, renal tissue MDA levels, and the protein expression levels of TNF-α, caspase-3, RIP1, RIP3, MLKL, CAMKII and CypD were significantly increased in the IR group compared to the control group (p < 0.05), Additionally, there were significant decreases in all the parameters in the IR + CsA group compared to those in the IR group (p < 0.05). Furthermore, histopathological analyses revealed significantly higher kidney injury scores in the IR group compared to the control group, and low dose CsA treatment improved the injury. A single low dose of CsA injection 15min before IR, demonstrated a protective effect on bilateral renal IR injury and a reduction in apoptotic and necroptopic markers which is resulted in improvement of renal functions.

TNF-alpha gene promoter's hypomethylation mediates a pro-inflammatory phenotype in peripheral blood monocytes from apical periodontitis individuals.

Epigenetic regulation of the key inflammatory genes plays a crucial role in controlling monocyte/macrophage-mediated local and systemic responses to bacterial challenges. However, it has not been addressed in apical periodontitis (AP). We aimed to explore the methylation pattern of the TNF-α gene promoter and its association with the inflammatory phenotype of peripheral blood monocytes from individuals with AP and controls. A cross-sectional study was conducted, including otherwise healthy individuals with AP (n = 25) and controls (n = 29). Monocytes were isolated from the volunteer's blood samples using a Ficoll gradient followed by negative immunoselection. RNA and DNA were extracted. The DNA methylation profiles of the TNF-α gene promoter region were analyzed using bisulfite sequencing PCR. The mRNA expression levels of DNA methyltransferases 3a (DNMT3a) and Ten Eleven Translocation enzymes 1(TET1) were assessed by qPCR. A fraction of primary monocytes was also cultured for 24 h, and the supernatant was collected to measure cytokine levels through a Luminex assay. Generalized structural equation models (GSEM) evaluated the association between AP, DNA methylation, and TNF-α protein expression controlled for potential covariates. Models included the effect of the methylation of TNF-α gene promoter as a mediator of the association between AP and TNF-α protein expression levels. Monocytes from AP individuals exhibited a heightened secretion of TNF-α and IL-1β and hypomethylation of the TNF gene promoter (p < .05). AP diagnosis was associated with the TNF-α gene promoter´s hypomethylated profile and enhanced pro-inflammatory cytokine levels, while lower methylation of the gene promoter region and -163 CpG single site mediated TNF-α overexpression (p < .05). DNA hypomethylation at the TNF-α gene mediates a proinflammatory phenotype in monocytes from AP patients, supporting a role in the systemic response.

Association between endometrial protein expression of IFN-γ and delayed conception after parturition in dairy cows.

The cytokine context present in the reproductive tract of cows is closely involved in normal uterine functions, including the estrous cycle and the establishment and maintenance of pregnancy. However, the roles of some cytokines in the uterus, and their relation with reproductive performance remain to be elucidated. Thus, this study aimed to examine the protein expression of several cytokines such as TNFα, IL-6, IL-8, IFNγ, IL-4, and TGF-β3 in endometrial biopsies previous to conception, to evaluate the possible association with delayed conception in dairy cows. Protein expression levels were evaluated by immunohistochemistry. Results showed that the protein expression levels of TNFα, IL-6, IL-4 and TGF-β3 were not associated with the parturition-conception interval, whereas the high protein expression levels of IFNγ were associated with the parturition-conception interval. Finally, the low protein expression of IL-8 showed a statistical tendency to be associated with delayed conception. This is the first report about the protein expression of IFN-γ in the endometrium of dairy cows and also, this cytokine could enhance the favorable conditions to achieve an early pregnancy.

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion.

To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1β and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1β in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1β in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

Examination of the Thermal Stability and Swallowing Safety of a Self-Developed Enteral Nutrition Thickening Agent

To experimentally validate the effects of a self-developed heat-stable thickening agent on the textual characteristics of enteral nutrition solutions of standard concentration and its applicability in improving dysphagia. A gradient of different doses of the self-developed thickening agent (1.0 g, 1.5 g, 2.0 g, 2.5 g, and3.0 g) and three commonly used commercial thickeners were mixed with 23.391 g of a complete nutrition formula powder dissolved in 85 mL of purified water to prepare 100 mL standard concentration nutrition solutions. The textual parameters (cohesiveness, viscosity, thickness, and hardness) of these nutrition solutions were measured using a texture analyzer at various temperature gradients (20 ℃, 40 ℃, 60 ℃, and 80 ℃) to compare their thermal stability. A dysphagia rat model was created via epiglottectomy to explore the effects of the thickener on lung tissue damage scores and levels of inflammatory markers. The rats were divided into a test intervention group, a positive control group, a negative control group, and a blank control group (no surgery and normal feeding after fasting for one day), with 15 rats in each group. After fasting for one day post-surgery, the test intervention group was fed with the standard concentration nutrition solution thickened with the self-developed thickener, while the positive control group was given a standard concentration nutrition solution thickened with product 3, and the negative control group was fed a normal diet. All groups were fed for two weeks with food dyed with food-grade green dye. General conditions, body mass, and food intake were observed and recorded. After two weeks, abdominal aorta blood was collected, and heart, liver, spleen, lung, and kidney tissues were harvested and weighed to calculate the lung tissue organ coefficient. The organ conditions were evaluated using routine H&E staining, and lung damage was semi-quantitatively analyzed based on the Mikawa scoring criteria. Blood supernatants were collected to measure the total serum protein and albumin levels to determine the nutritional status of the rats. The expression of IL-6 and TNF-α genes in lung tissues was measured by RT-qPCR. IL-6 and TNF-α protein expression levels in lung tissues, lung tissue homogenate, and serum were measured by ELISA. The aspiration incidence rate was calculated. Within the dosage range of 1.0 g to 3.0 g, the self-developed thickener in the test samples exhibited superior thermal stability in cohesiveness compared to the three commercially available thickeners, with a statistically significant difference (P<0.01). The differences in the thermal stability of viscosity and hardness between the self-developed thickener and the three commercially available thickeners were not statistically significant. The viscosity stability was optimal for the self-developed thickener, followed by the commercially available thickeners 1 and 3, with thickeners 2 being the least stable, though the differences were not statistically significant (P>0.05). Product 1 showed the best thermal stability in thickness, followed by the self-developed thickener and product 2, while the product 3 exhibited the worst performance, with the difference being statistically significant (P<0.01). The self-developed thickener had the best thermal stability in hardness at temperatures ranging from 20℃ to 80 ℃, followed by products 1 and 2, with product 3 being the least stable. However, the differences were not statistically significant (P>0.05). Animal experiment results indicated that the body weight gain in the positive control group and the test intervention group was lower than that in the blank and negative control groups (P<0.01). The spleen coefficient of the intervention group was lower than that of the positive control group and the blank control group (P<0.01), while the heart, liver, and kidney coefficients were lower than those of the blank control group (P<0.01). The differences in the lung coefficient of the intervention group and those of the other three groups were no statistically significant. Levels of TP and ALB in the test intervention group, the positive control group, and the negative control group were all lower than those in the blank control group, with statistically significant differences (P<0.01). ELISA results showed that serum IL-6 levels in the blank and test intervention groups were lower than those in the negative and positive control groups (P<0.05), while the difference in the other indicators across the four groups were not statistically significant (P>0.05). There were no statistically significant differences among the four groups in terms of lung tissue damage pathology scores, or in the levels of IL-6 and TNF-α gene expression in lung tissues. The aspiration incidence rate was 0% in all groups. The self-developed enteral nutrition thickening agent demonstrated excellent thermal stability and swallowing safety. Further research to explore its application in patients with dysphagia is warranted.

Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism

Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism

Identification and preliminary validation of differently expressed genes as candidate biomarkers associated with atherosclerosis

ObjectiveAtherosclerosis (AS) is the primary cause of deadly cardio-cerebro vascular diseases globally. This study aims to explore the key differentially expressed genes (DEGs), potentially serving as predictive biomarkers for AS. MethodsMicroarray datasets were retrieved from the GEO database for DEGs and DE-miRNAs identification. Then biological function of DEGs were elucidated based on gene ontology (GO) and KEGG pathway enrichment analysis. The protein–protein interaction (PPI) network and DEGs-DE-miRNAs network were constructed, with emphasis on hub DEGs selection and their interconnections. Additionally, receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic precision of hub DEGs for AS. More importantly, an AS Syrian Golden hamster model was established to validate the expression levels of hub DEGs in AS. ResultsA total of 203 DEGs and 10 DE-miRNAs were screened, with six genes were chosen as hub DEGs. These DEGs were significantly enriched in AS-related biological processes and pathways, such as immune and inflammatory response, cellular response to IL-1 and TNF, positive regulation of angiogenesis, Type I diabetes mellitus, Cytokine-cytokine receptor interaction, TLR signaling pathway. Also, these DEGs and DE-miRNAs formed a closely-interacted DE-miRNAs - DEGs - KEGG pathway network. Besides, hub DEGs presented promising diagnostic potential for AS (AUC: 0.781 ∼ 0.887). In addition, the protein expression levels of TNF-α, CXCL8, CCL4, IL-1β, CCL3 and CCR8 were significantly increased in AS group Syrian Golden hamsters. ConclusionThe identified candidate genes TNF, CXCL8, CCL4, IL1B, CCL3 and CCR8 may have the potential to serve as prognostic biomarker in diagnosing AS.

The effects of IGF-1 and IGFBP-2 treatments on the atherosclerosis in the aorta and the coronary arteries of the high cholesterol diet-fed rabbits

Several studies have demonstrated suppression of aortic atherosclerosis by insulin like growth factor-1 (IGF-1) in hypercholesterolemic rabbits. Though a recent study has reported that IGF-1 exerts anti-atherogenic effects in coronary arteries, the mechanisms of IGF-1 in coronary arteries need to be further verified. Studies about insulin like growth factor binding protein-2 (IGFBP-2) in atherosclerosis are rarely. The objective of this study is to examine the effects of IGF-1 and IGFBP-2 on the atherosclerosis development in the aorta and coronary arteries of the high-cholesterol diet (HCD)-fed rabbits. New Zealand white rabbits were fed either normal chow (n = 5) or a diet containing 1.0 % cholesterol (n = 18) for 12 weeks. Cholesterol-fed rabbits were given IGF-1 or IGFBP-2 or saline intravenously (each n = 6) for 10 weeks. The results revealed that IGF-1 decreased total cholesterol (TC) and low-density lipoprotein (LDL) levels (p < 0.05), whereas IGFBP-2 did not. IGF-1 significantly attenuated atherosclerotic lesions and reduced accumulated macrophages within the coronary artery plaques, whereas IGFBP-2 deteriorated these changes. Moreover, IGF-1 reduced serum platelet-activating factor acetylhydrolase levels, C reactive protein (CRP), and inhibited the protein expression levels of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). IGFBP-2 elevated serum 8-hydroxy-2′-deoxyguanosine levels, CRP, and promoted the protein expression levels of TNF-α and IL-6. In conclusion, IGF-1 can substantially suppress plaque formation in coronary arteries with a marked inhibition of macrophage accumulation likely via its anti-inflammatory properties, whereas IGFBP-2 plays an opposite effect on atherosclerosis. The present study highlighted a theoretical basis for pharmacological treatment of atherosclerosis.

Integrating Network Pharmacology and an Experimental Model to Investigate the Effect of Zhenwu Decoction on Doxorubicin-Induced Heart Failure.

Doxorubicin-induced heart failure is a clinical problem that needs to be solved urgently. Previous studies have confirmed that Zhenwu Decoction, a traditional Chinese medicine compound, can effectively improve chronic heart failure. However, its interventional effect on Doxorubicin-induced heart failure has not yet been investigated. In this study, we investigated the therapeutic effect and potential mechanism of Zhenwu Decoction on Doxorubicin-induced heart failure through animal experiments and network pharmacology. The study aimed to investigate the therapeutic effect and potential mechanism of Zhenwu Decoction (ZWD) on Doxorubicin-induced heart failure. A heart-failure mouse model was established in 8-week-old male C57/BL6J mice using Doxorubicin, and the mice were then treated with ZWD for a 4-week period. Firstly, network pharmacology was conducted to explore the potential active components and molecular mechanisms of ZWD on Doxorubicin-induced heart failure. Next, we conducted an in vivo study on the effect of ZWD on Doxorubicin-induced heart failure. After the intervention, the cardiac function and levels of cardiac function injury marker in serum were measured to evaluate the therapeutic effect of ZWD on cardiac function. Then HE staining and Masson staining were used to evaluate the effect of ZWD on myocardial pathology, and biochemical method was used to detect the effect of ZWD on total antioxidant capacity and inflammation, and finally, Western blot was used to detect TGFβ, Smad-3, and collagen I protein expression levels to evaluate its effect on myocardial fibrosis. In Doxorubicin-induced heart failure mice, ZWD improved cardiac function and reduced the levels of CK-MB, NT-proBNP, and BNP in the serum, improved myocardial pathology, and reduced TGFβ, Smad-3 and collagen I protein expression levels to improve myocardial fibrosis. Network pharmacological analysis showed that ZWD has 146 active ingredients and 248 candidate targets. Moreover, 2,809 genes were found to be related to Doxorubicin-induced heart failure, and after screening, 74 common targets were obtained, mainly including IL-6, AKT1, caspase-3, PPARG, PTGS2, JUN, HSP90AA1, and ESR1. KEGG analysis confirmed that PI3K/AKT and IL-6/NF-κB signaling pathways were the two main pathways underlying the cardioprotective effects of ZWD. Finally, in vivo experiments showed that ZWD improved the total antioxidant capacity, reduced the SOD level, increased the protein expression of PI3K, Akt, Bcl-2, Bax, and caspase-3, reduced the levels of TNF-α, IL-6, and IL-1β, and decreased the NF-κB p65, IL-6, and TNF-α protein expression levels. In Doxorubicin-induced heart-failure mice, Zhenwu Decoction improved the cardiac function and myocardial pathology, and improved myocardial fibrosis through the TGFβ/Smad-3 signaling pathway. According to the prediction of network pharmacology, in vivo experiments demonstrated that Zhenwu Decoction can improve the oxidative stress response, improve myocardial cell apoptosis through the PI3K/AKT signaling pathway, and improve myocardial inflammation by reducing the levels of inflammatory factors and by reducing the protein expression of NF-κB p65, IL-6, and TNF-α.

Fullerene C60 Attenuates Heart Tissue Inflammation by Modulating COX-2 and TNF-Alpha Signaling Pathways in DMBA Induced Breast Cancer in Rats.

The present study aimed to investigate the therapeutic effect of fullerene C60 nanoparticle against heart tissue damage caused by 7,12-dimethylbenz [a] anthracene (DMBA) in female rats. Female Wistar albino rats, 8weeks old (n = 60) weighing around (150 ± 10g) were used for the study. These rats were divided into 4 groups and each group included 15 rats. Groups: (i) Control Group: Fed with standard diet; (ii) C60 Group: C60 (1.7mg/kg bw, oral gavage); (iii) DMBA Group: DMBA (45mg/kg bw, oral gavage); (iv) C60 and DMBA Group: C60 (1.7mg/kg bw, oral gavage) and DMBA (45mg/kg bw, oral gavage) group. Malondialdehyde (MDA) analysis, catalase activity (CAT), and glutathione (GSH) in heart tissue were determined by spectrophotometer. In addition, heart tissue DNA damage was investigated. Caspase-3, p53, HO-1, COX-2, and TNF-α protein expression levels in heart tissue were determined by western blotting. As a result, Caspase-3, p53, HO-1 protein expression, GSHlevels and CAT activity increased, COX-2, TNF-α protein expression, and MDA levels were significantly decreased in the C60 + DMBAgroup compared to the DMBAgroup. Therefore, the fullerene C60 nanoparticle may be a promising and effective therapy for the treatment of heart diseases associated with inflammation.

Xinyang Tablet attenuates chronic hypoxia-induced right ventricular remodeling via inhibiting cardiomyocytes apoptosis

BackgroundHypoxia-induced pulmonary hypertension (HPH) is one of the fatal pathologies developed under hypobaric hypoxia and eventually leads to right ventricular (RV) remodeling and RV failure. Clinically, the mortality rate of RV failure caused by HPH is high and lacks effective drugs. Xinyang Tablet (XYT), a traditional Chinese medicine exhibits significant efficacy in the treatment of congestive heart failure and cardiac dysfunction. However, the effects of XYT on chronic hypoxia-induced RV failure are not clear.MethodsThe content of XYT was analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS). Sprague–Dawley (SD) rats were housed in a hypobaric chamber (equal to the parameter in altitude 5500 m) for 21 days to obtain the RV remodeling model. Electrocardiogram (ECG) and hemodynamic parameters were measured by iWorx Acquisition & Analysis System. Pathological morphological changes in the RV and pulmonary vessels were observed by H&E staining and Masson’s trichrome staining. Myocardial apoptosis was tested by TUNEL assay. Protein expression levels of TNF-α, IL-6, Bax, Bcl-2, and caspase-3 in the RV and H9c2 cells were detected by western blot. Meanwhile, H9c2 cells were induced by CoCl2 to establish a hypoxia injury model to verify the protective effect and mechanisms of XYT. A CCK-8 assay was performed to determine the viability of H9c2 cells. CoCl2-induced apoptosis was detected by Annexin-FITC/PI flow cytometry and Hoechst 33,258 staining.ResultsXYT remarkably improved RV hemodynamic disorder and ECG parameters. XYT attenuated hypoxia-induced pathological injury in RV and pulmonary vessels. We also observed that XYT treatment decreased the expression levels of TNF-α, IL-6, Bax/Bcl-2 ratio, and the numbers of myocardial apoptosis in RV. In H9c2 myocardial hypoxia model, XYT protected H9c2 cells against Cobalt chloride (CoCl2)-induced apoptosis. We also found that XYT could antagonize CoCl2-induced apoptosis through upregulating Bcl-2, inhibiting Bax and caspase-3 expression.ConclusionsWe concluded that XYT improved hypoxia-induced RV remodeling and protected against cardiac injury by inhibiting apoptosis pathway in vivo and vitro models, which may be a promising therapeutic strategy for clinical management of hypoxia-induced cardiac injury.

Ulinastatin ameliorates preeclampsia induced by N(gamma)-nitro-l-arginine methyl ester in a rat model via inhibition of the systemic and placental inflammatory response.

The pathogenesis of preeclampsia (PE) is associated with inflammation and endothelial damage. Ulinastatin (UTI) mainly inhibits proteolytic activity and significantly reduces the release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) from macrophages. It also ameliorates vascular endothelial damage in pathological conditions. Hence, we investigated the effects of UTI in a rat model of PE induced using N(gamma)-nitro-l-arginine methyl ester (L-NAME). Although inducing PE in a rat model, 5000 U/kg of UTI were injected daily. Systolic blood pressure (SBP) and protein levels in the urine were measured. Renal function, and serum concentrations of TNF-α, IL-6, placental growth factor (PLGF), and von Willebrand factor (vWF) were evaluated. The number and weight of live fetuses as well as the weight of placentas were measured. Placentas were collected for western blot and pathological analysis. UTI slightly ameliorated proteinuria and the increases in SBP, blood urea nitrogen (BUN), and serum creatinine. Furthermore, UTI improved serum and placental protein expression levels of TNF-α, IL-6, vWF, and PLGF. Pathological analysis revealed that vascular density and blood flow perfusion was enhanced, vessel wall thickening and neutrophil infiltration were diminished, and the weight and number of live fetuses as well as the weight of the placentas were improved with UTI. Preventive use of UTI in the PE rat model induced by L-NAME partially alleviated hypertension, proteinuria, and impaired renal function; improved fetal growth restriction; diminished vascular endothelial injury; and ameliorated placental vasculogenesis abnormality and malperfusion by inhibiting the systemic and placental inflammatory response, suggesting that UTI is a potential drug for PE prevention or treatment.

Neuroprotective effects of etanercept on diabetic retinopathy via regulation of the TNF-α/NF-κB signaling pathway

Purpose: To study the influence of etanercept on diabetic retinopathy in rats via tumor necrosis factor alpha (TNF-α)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. &#x0D; Methods: Thirty-six Sprague-Dawley (SD) rats were randomly divided into normal, model and etanercept groups. The expression of Caspase-3 was determined by immunohistochemistry, while the relative protein and mRNA expression levels of TNF-α and NF-κB were determined by Western blotting and quantitative polymerase chain reaction, respectively. Besides, the contents of TNF-α and interleukin-1 beta (IL-1β) were evaluated using enzyme-linked immunosorbent assay (ELISA), while cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). &#x0D; Results: Immunohistochemical studies showed that the mean optical density of tissues positive for caspase-3 in both the model and etanercept groups were significantly higher than in the normal group (p &lt; 0.05), while the mean optical density in the etanercept group was significantly lower than that in the model group (p &lt; 0.05). The protein expression levels of TNF-α and NF-κB in the etanercept group were significantly lower than those in the model group (p &lt; 0.05). Furthermore, mRNA expressions of TNF-α and NF-κB declined in the etanercept group (p &lt; 0.05); in addition, TNF-α, and IL-1β levels in the etanercept group were lower than in the model group (p &lt; 0.05). Cell apoptosis in the etanercept group was also lower than in the model group. &#x0D; Conclusion: Etanercept suppresses TNF-α/NF-κB signaling pathway thereby repressing inflammation and cell apoptosis in diabetic retinopathy rats. Therefore, etenercept’s neuroprotective effect may potentially be useful in developing a suitable therapy for diabetic neuropathy.

Kaempferol Inhibits Dry Eye-Related Corneal Inflammation In Vitro Via the p38 Pathway

ObjectiveThe purpose of this study is to investigate the effect of kaempferol (KAE), an effective constituent of Buddleja officinalis, on human corneal epithelial cells (HCECs) under a hyperosmolar environment. Methods HCECs were cultured in DMEM/F12 medium and allocated into four groups: normal, model, vehicle, and KAE. Ki67 staining was used to detect HCEC proliferation; TdT-mediated dUTP Nick-End Labeling staining was used to detect HCEC apoptosis; quantified real-time PCR was used to investigate the mRNA relative expression levels of TNF-α, IL-6, and p38. and Western blot was used to reveal the protein expression levels of TNF-α, IL-6, and p38. Results Compared with those in the normal group, the proliferation ability of HCECs was significantly inhibited and the apoptosis rate of HCECs was notably increased in the model group; but KAE could effectively improve HCEC proliferation and reduce HCEC apoptosis. Compared with those in the normal group, the mRNA relative expressions and protein levels of TNF-α, IL-6, and p38 were significantly increased in the model group, which were effectively decreased after KAE intervention. Conclusion KAE could promote HCEC proliferation and reduce cell apoptosis, as well as inhibit the expression of inflammatory factors in a hypertonic-induced HCEC model via the p38MAPK pathway.

Ginsenoside Rb1 attenuates lipopolysaccharide-induced chronic neuroinflammation in mice by tuning glial cell polarization

To evaluate whether ginsenoside Rb1 (Rb1) can attenuate lipopolysaccharide (LPS)-induced chronic neuroinflammation in mice and to explore its relationship with glial cell polarization. Intraperitoneal injection with an escalating dose of LPS was used to establish a chronic neuroinflammation model in mice. Once LPS was initiated, 10 or 20 mg/kg Rb1, or sterile saline, was administered for 14 consecutive days. Open field test and beam walking test were used to monitor the changes in behavior. The concentration of cytokines in the serum and brain were used to monitor the systemic inflammation and neuroinflammation, respectively. Molecules specific to each glial cell phenotype were used to investigate glial cell polarization. Mice in the LPS group had reduced spontaneous activities and impaired beam walking performance. Rb1 obviously eased LPS-induced behavior disturbances. Regarding the levels of serum cytokines, both tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased, while interleukin-10 (IL-10) and transforming growth factor β (TGF-β) remarkably decreased after LPS treatment (all P < .001). Rb1 treatment significantly attenuated LPS-induced serum cytokine changes (all P < .05). The results of quantitative polymerase chain reaction and western blotting showed that the mRNA and protein expression levels of TNF-α and complement component 3 (C3) in the brain were significantly increased after LPS treatment (all P < .01). Rb1 treatment significantly inhibited LPS-induced inflammation in the brain (all P < .05). Glial cell polarization analysis showed that M1 and M2 microglia, and A1 astrocytes increased following LPS treatment, while A2 astrocytes decreased. Rb1 treatment reduced M1 and M2 microglia, and A1 astrocytes, and significantly increased A2 astrocytes. Rb1 can attenuate chronic neuroinflammation induced by LPS in mice, which may be partially attributable to its fine tuning of microglia and astrocyte polarization. Rb1 has potential value for treating neurodegenerative diseases.

Luteolin is an Effective Component of Platycodon grandiflorus in Promoting Wound Healing in Rats with Cutaneous Scald Injury.

BackgroundPlatycodon grandiflorus could significantly improve the pathological results of cutaneous scald injury, reduce the release of inflammatory factors and promote angiogenesis. This study investigated the wound healing effect of luteolin, an active component of P. grandiflorus, on induced cutaneous scald injury in Sprague-Dawley (SD) rats.MethodsThe protein expression levels of TNF-α and IL-6 were detected by ELISA. QRT-PCR was adopted to detect the expression of TGF-β1 and VEGF. Histopathological changes of scald wounds were analyzed by hematoxylin-eosin staining. Cell viability and migration ability were detected by CCK-8 assay and scratch assay.ResultsBoth in vivo and in vitro experiments showed that luteolin promoted wound healing of cutaneous scald injury. Gene Oncology (GO) functional analysis and rescue experiments showed that endothelial nitric oxide synthase 3 (NOS3) was the critical target of luteolin in treating cutaneous scald.ConclusionThis study demonstrated that luteolin is an effective component of P. grandiflorus and is effective in the treatment of cutaneous scald injury.

Early-Life Nutrition Interventions Improved Growth Performance and Intestinal Health via the Gut Microbiota in Piglets.

Intestinal infections in piglets are the main causes of morbidity before and after weaning. Studies have not explored approaches for combining pre-weaning and post-weaning nutritional strategies to sustain optimal gut health. The current study thus sought to explore the effects of early-life nutrition interventions through administration of synthetic milk on growth performance and gut health in piglets from 3 to 30 days of age. Twelve sows were randomly allocated to control group (CON) and early-life nutrition interventions group (ENI). Piglets were fed with the same creep diet from 7 days of age ad libitum. Piglets in the ENI group were provided with additional synthetic milk from Day 3 to Day 30. The results showed that early-life nutrition interventions improved growth performance, liver weight, spleen weight, and reduced diarrhea rate of piglets after weaning (P < 0.05). Early-life nutrition interventions significantly upregulated expression of ZO-1, Occludin, Claudin4, GALNT1, B3GNT6, and MUC2 in colonic mucosa at mRNA level (P < 0.05). Early-life nutrition interventions reduced activity of alkaline phosphatase (AKP) in serum and the content of lipopolysaccharides (LPS) in plasma (P < 0.05). The number of goblet cells and crypt depth of colon of piglets was significantly higher in piglets in the ENI group relative to that of piglets in the CON group (P < 0.05). The relative mRNA expression levels of MCP-1, TNF-α, IL-1β, and IL-8, and the protein expression levels of TNF-α, IL-6, and IL-8 in colonic mucosa of piglets in the ENI group were lower compared with those of piglets in the CON group (P < 0.05). Relative abundance of Lactobacillus in colonic chyme and mucosa of piglets in the ENI group was significantly higher relative to that of piglets in the CON group (P < 0.05). Correlation analysis indicated that abundance of Lactobacillus was positively correlated with the relative mRNA expression levels of ZO-1, Claudin4, and GALNT1, and it was negatively correlated with the level of MCP-1 in colonic chyme and mucosa. In summary, the findings of this study showed that early-life nutrition interventions improved growth performance, colonic barrier, and reduced inflammation in the colon by modulating composition of gut microbiota in piglets. Early-life nutrition intervention through supplemental synthetic milk is a feasible measure to improve the health and reduce the number of deaths of piglets.

Elafin-expressing probiotic Escherichia coli Nissle 1917 protects against experimental colitis

Objective: To construct the gene modified probiotic Escherichia coli nissle1917 (EcN) which can express human Elafin protein and to explore its protective effect on the acute colitis in mice. Methods: The recombinant plasmid with human Elafin gene was constructed and then transferred to EcN. Western blot results confirmed that the engineered probiotic expressed Elafin successfully in vitro. C57/BL6J mouse was used in this study and were randomly divided into 4 groups according to different treatment: PBS gavage (PBS group); DSS administrated (DSS group); DSS administrated with wild-type EcN (EcN-WT) gavage (EcN-WT group); DSS administrated with EcN-Elafin gavage (EcN-Elafin group). Body weight and disease activity index (DAI) were measured every day. The length of mice colons in each group were measured after euthanasia. The degree of inflammation of intestinal mucosa in each group was measured through histopathological scoring. The proportion of neutrophils and macrophages infiltrated into colon lamina propria was detected by flow cytometry. The protein expression levels of pro-inflammatory cytokines TNF-α, IL-6 and chemokine CXCL-1 in colonic tissue were quantified by enzyme-linked immunosorbent assay. Results: Elafin protein could be detected in the supernatant of EcN-Elafin culture medium and EcN-Elafin homogenates. Compared with DSS group, the weight loss and DAI score of EcN-Elafin group and EcN-WT group were both significantly improved. The colon length of EcN-Elafin group was significantly longer than that of DSS group. The histological score of colitis in EcN-Elafin group was significantly lower than that in DSS group (5.3±2.3 vs 9.3±1.4, P<0.05). In EcN-Elafin group, the proportion of neutrophils[(8.65±1.49)% vs (17.60±2.16)%, P<0.01]and macrophages[(3.79±0.26)% vs (5.73±0.45)%, P<0.01]infiltrated into the colon lamina propria was significantly decreased compared with DSS group. The protein expression levels of TNF-α, IL-6 and CXCL-1 in EcN-Elafin group and EcN-WT group were significantly lower than those in DSS group. Conclusion: Elafin-expressing EcN can protect against DSS-induced acute colitis in mice and may have provided an effective and cost-efficient method for the treatment of IBD.

Pyruvate kinase M2 (PKM2) improve symptoms of post-ischemic stroke depression by activating VEGF to mediate the MAPK/ERK pathway.

PurposeTo evaluate and identify the effects and explore the mechanisms of pyruvate kinase M2 (PKM2) on stroke‐induced post stroke depression (PSD).MethodsRats were separated into six different groups, including sham + saline, Stroke + saline, PSD + saline, PSD + recombinant pyruvate kinase M2 (rPKM2) (112 ng/kg), PSD + rPKM2 (224 ng/kg), and PSD + rPKM2 (224 ng/kg) + bevacizumab. Then, the body weight, sucrose preference rate, immobility time, horizontal movement, and vertical movement were determined to evaluate the effect of PKM2 on improving the depressive behavior of PSD rats. Subsequently, the proliferation of oligodendrocytes in subventricular zone (SVZ) of rats in each group was examined by western blot and immunofluorescent staining. Furthermore, the mRNA and protein expression levels of TNF‐α, IL‐6, and IL‐1β were also detected by qPCR and ELISA to verify the anti‐inflammatory effects of PKM2 on PSD rats. In addition, the protein expression levels of MDA, LDH, and NO were tested to reveal that PKM2 can reduce oxidative stress in PSD rats. The western blot and IHC assays were employed to examine the protein expression levels of VEGF, PKM2, and ERK in PSD rats.ResultsIn this study, the results showed that PKM2 can improve the depressive behavior and proliferation of oligodendrocytes in PSD rats. In addition, PKM2 has anti‐inflammatory and anti‐oxidative stress effects on PSD rats. Meanwhile, PKM2 activated the expression level of VEGF/MAPK/ERK pathway.ConclusionPKM2 improves symptoms of post‐ischemic stroke depression by activating VEGF‐mediated MAPK/ERK pathway.

Mechanisms of sodium butyrate inhibition of microglia inflammatory activation in hippocampus via Toll-like receptor 4/nuclear factor-κB p65 pathway

To investigate the effects of sodium butyrate (NaB) on long-term anxiety like behavior and inflammatory activation of microglia in the hippocampus of sepsis-associated encephalopathy (SAE) mice. (1) Animal experiment: fifty C57BL/6 mice aged 6-8 weeks were randomly divided into Sham group (only the cecum was found by laparotomy without perforation or ligation), and SAE model group caused by cecal ligation and puncture (CLP; SAE model group, the cecum was found by laparotomy and perforated after ligation. The open field test indicated that the ability of independent exploration decreased and showed anxiety like behavior, which proved that the SAE model was successfully replicated) and NaB pretreatment group was established (NaB was administered at a dose of 500 mg×kg-1×d-1 for 3 days before modeling, and the same dose once a day for 3 days after modeling). Open field test was used to detect the anxiety like behavior of mice at 7 days. The protein expressions and content changes of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in hippocampus of mice at 1 day and 3 days after operation were detected by Western blotting and enzyme linked immunosorbent assay (ELISA). Immunofluorescence staining was used to observe microglia labeled protein ionized calcium bindingadaptor molecule-1 (Iba-1) and TNF-α protein co localization. (2) Cell experiment: mouse microglia cell line BV-2 microglia were divided into blank control group, lipopolysaccharide (LPS) group (cells were treated with 1 mg/L LPS), and NaB treatment group (cells were treated with 1 mg/L LPS+5 mmol/L NaB). The protein expressions of IL-1β, TNF-α, Toll-like receptor 4 (TLR4), phosphorylated nuclear factor-κB p65 (p-NF-κB p65), nuclear factor-κB p65 (NF-κB p65) and NF-κB inhibitor protein-α (IκB-α) were detected by Western blotting. The expressions of Iba-1 and TNF-α in each group were observed by immunofluorescence. (1) Animal experiment: compared with the Sham group, the distance and duration of movement in the central area, the total distance moved of mice decreased 7 days after the establishment of SAE model group were decreased [distance of movement in the central area (mm): 13.45±3.97 vs. 161.44±27.00, duration of movement in the central area (s): 1.82±0.58 vs. 13.45±2.17, the total distance moved (mm): 835.01±669.67 vs. 2 254.51±213.45, all P < 0.05]. In the hippocampus tissues of mice, a large number of nerve nuclei were pyknotic and deeply stained, and the arrangement of nerve cells was disordered. The cell bodies of microglia in mouse hippocampus increased significantly. The number of positive cells of Iba-1/TNF-α (Iba-1+/TNF-α+) increased significantly. The contents and protein expression of proinflammatory factors TNF-α, IL-1β in hippocampal homogenate supernatant 3 days after operation in SAE model group were significantly higher than those in Sham group [TNF-α (ng/L): 119.17±18.40 vs. 90.18±21.17, IL-1β (ng/L): 407.89±70.64 vs. 313.69±34.63; TNF-α/GAPDH: 1.42±0.50 vs. 0.80±0.08, IL-1β/GAPDH: 1.27±0.22 vs. 0.85±0.25, all P < 0.05]. After intragastric administration of NaB, the distance and duration of movement in the central area of mice were significantly higher than those in SAE model group [distance of movement in the central area (mm): 47.39±15.63 vs. 13.45±3.97, duration of movement in the central area (s): 6.12±1.87 vs. 1.82±0.58, all P < 0.05]. There was no significant change in the total distance moved (mm: 1 550.59±1 004.10 vs. 835.01±669.67, P > 0.05). The pyknosis and deep staining of nerve nuclei in mice were significantly less than those in SAE model group. The number of Iba-1+/TNF-α+ positive cells decreased significantly. The contents and protein expression levels of proinflammatory factors TNF-α, IL-1β in hippocampal homogenate supernatant 3 days after operation were significantly lower than those in SAE model group [TNF-α (ng/L): 64.95±9.10 vs. 119.17±18.40, IL-1β (ng/L): 311.94±69.92 vs. 407.89±70.64; TNF-α/GAPDH: 1.02±0.36 vs. 1.42±0.50, IL-1β/GAPDH: 0.86±0.20 vs. 1.27±0.22, all P < 0.05]. (2) Cell experiment: after LPS intervention, the fluorescence intensity of TNF-α in BV-2 cells was significantly enhanced, the protein expression levels of TNF-α, IL-1β, TLR4 and p-NF-κB p65 protein increased (TNF-α/GAPDH: 0.39±0.06 vs. 0.20±0.02, IL-1β/GAPDH: 0.27±0.03 vs. 0.19±0.01, TLR4/GAPDH: 0.55±0.12 vs. 0.33±0.09, p-NF-κB p65/NF-κB p65: 0.55±0.05 vs. 0.29±0.04, all P < 0.05), the expression level of IκB-α was lower than that in the control group(IκB-α/GAPDH: 0.54±0.06 vs. 0.81±0.03, P < 0.05). After NaB treatment, the fluorescence intensity of TNF-α in BV-2 cells was decreased. The protein expression levels of TNF-α, IL-1β, TLR4 and p-NF-κB p65 protein were significantly lower than that of LPS model group (TNF-α/GAPDH: 0.26±0.02 vs. 0.39±0.06, IL-1β/GAPDH: 0.11±0.04 vs. 0.27±0.03, TLR4/GAPDH: 0.28±0.14 vs. 0.55±0.12, p-NF-κB p65/NF-κB p65: 0.29±0.01 vs. 0.55±0.05, all P < 0.05), the protein expression level of IκB-α was significantly higher than that in the LPS group (IκB-α/GAPDH: 0.75±0.01 vs. 0.54±0.06, P < 0.05). NaB could antagonism the TLR4 activation induced by LPS, thus inhibiting p-NF-κB p65 nuclear transcription and IκB-α degradation. It can reduce microglia activation and secretion of inflammatory factors, and finally improve the inflammation in the hippocampus of septic mice and long-term anxiety like behavior.