Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate-biotin Nick Research Articles

Multi-parametric MRI assessment of melatonin regulating the polarization of microglia in rats after cerebral ischemia/reperfusion injury

ObjectivesMulti-parametric magnetic resonance imaging (MRI) can provide comprehensive and valuable information for precise diagnosis and treatment evaluation of a number of diseases. In this study, the neuroprotective effects of melatonin (Mel) on a rat model of cerebral ischemia/reperfusion injury (CIRI) were assessed by multi-parametric MRI combined with histopathological techniques for longitudinal monitoring of the lesion microenvironment. MethodsSixty Sprague Dawley (SD) rats were randomly divided into three groups: the Sham, CIRI and CIRI + Mel groups. At multiple time points after ischemia, MRI scanning was performed on a 7.0 Tesla MRI scanner. Multi-parametric MRI includes T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and chemical exchange saturation transfer (CEST)-MRI. CEST effects were calculated by the Lorentzian difference method, 3.5 ppm indicates amide protons of mobile proteins/peptide (Amide-CEST) and 2.0 ppm indicates amine protons (Guan-CEST). Multiple histopathological techniques were used to examine the histopathological changes and explore the therapeutic effects of Mel. ResultsT2WI and DWI-MRI could localize the infarct foci and areas in CIRI rats, which was further validated by staining, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining. After Mel treatment, T2WI and DWI-MRI showed smaller infarct volume, and neurons displayed improved morphology with less apoptosis rates. Notably, Amide-CEST and Guan-CEST signal decreased as early as 2 h after CIRI (all P <0.001), reflecting the change of pH after ischemia. After Mel treatment, both Amide-CEST and Guan-CEST signal increased in ischemic cortex and striatum compared with control group (all P < 0.001). The immunofluorescence staining and western blotting analysis suggested the expression of M2 microglia increased after Mel treatment; While，after Mel treatment the inflammatory factor interleukin-1β (IL-1β) decreased compared with control CIRI rats. ConclusionsMulti-parametric MRI was shown to be an effective method to monitor the brain damage in a rat model of CIRI and assess the therapeutic effects of Mel treatment. Amide-CEST and Guan-CEST were especially sensitive to the changes in brain microenvironment during the early stage after CIRI. Furthermore, the neuroprotective effect of Mel treatment is associated with its promotion of the microglia polarized to M2 type in CIRI rats.

Tilianin alleviates lipid deposition and fibrosis in mice with nonalcoholic steatohepatitis by activating the PPARα/Nnat axis.

The understanding and treatment of nonalcoholic steatohepatitis (NASH) are still very limited. This study reports the therapeutic effect of tilianin on mice with NASH and further explores its possible molecular mechanisms. A mice model of NASH was established using low-dose streptozotocin combined with a high-fat diet and tilianin treatment. Liver function was assessed by determining serum aspartate aminotransferase and alanine aminotransferase in serum. Interleukin (IL)-1β, IL-6, transforming growth factor β1 (TGF-β1), and tumor necrosis factor α (TNF-α) levels in serum were determined. Hepatocyte apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling staining. Oil Red O staining and boron dipyrrin staining were used to determine lipid deposition in liver tissues. Masson staining was used to evaluate liver fibrosis, and immunohistochemistry and western blot analysis were used to determine the expression of target proteins. Tilianin treatment significantly ameliorated liver function, inhibited hepatocyte apoptosis, and reduced lipid deposition and liver fibrosis in mice with NASH. The expression of neuronatin (Nnat) and peroxisome proliferator-activated receptor (PPAR) α was upregulated, whereas that of sterol regulatory element-binding protein 1 (SREBP-1), TGF-β1, nuclear factor (NF)-κB p65, and phosphorylated p65 was downregulated in the liver tissues of mice with NASH after tilianin treatment. The above effects of tilianin were significantly reversed after Nnat knock-down, but its effect on PPARα expression was unaffected. Thus, the natural drug tilianin shows potential in treatig NASH. Its mechanism of action may be related to the targeted activation of PPARα/Nnat, thereby inhibiting the activation of the NF-κB signaling pathway.

Characterization of the Developing Lacunocanalicular Network During Fracture Repair.

ABSTRACTFracture repair is a normal physiological response to bone injury. During the process of bony callus formation, a lacunocanalicular network (LCN) is formed de novo that evolves with callus remodeling. Our aim was the longitudinal assessment of the development and evolution of the LCN during fracture repair. To this end, 45 adult wild‐type C57BL/6 mice underwent closed tibial fracture surgery. Fractured and intact contralateral tibias were harvested after 2, 3, and 6 weeks of bone healing (n = 15/group). High‐resolution micro–computed tomography (μCT) and deconvolution microscopy (DV) approaches were applied to quantify lacunar number density from the calluses and intact bone. On histological sections, Goldner's trichrome staining was used to assess lacunar occupancy, fluorescein isothiocyanate staining to visualize the canalicular network, and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) staining to examine osteocyte apoptosis. Analysis of μCT scans showed progressive decreases in mean lacuna volume over time (−27% 2–3 weeks; −13% 3–6 weeks). Lacunar number density increased considerably between 2 and 3 weeks (+156%). Correlation analysis was performed, showing a positive linear relationship between canalicular number density and trabecular thickness (R 2 = 0.56, p < 0.001) and an inverse relationship between mean lacuna volume and trabecular thickness (R 2 = 0.57, p < 0.001). Histology showed increases in canalicular number density over time (+22% 2–3 weeks, +51% 3–6 weeks). Lacunar occupancy in new bone of the callus was high (>90%), but the old cortical bone within the fracture site appeared necrotic as it underwent resorption. In conclusion, our data shows a progressive increase in the complexity of the LCN over time during fracture healing and demonstrates that this network is initiated during the early stages of repair. Further studies are needed to address the functional importance of osteocytes in bone healing, particularly in detecting and translating the effects of micromotion in the fracture. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Sevoflurane ameliorates adriamycin-induced myocardial injury in rats through the PI3K/Akt/GSK-3β pathway.

The aim of this study was to explore the effects of sevoflurane (SEV) pretreatment on Adriamycin (ADR)-induced myocardial injury through the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) pathway. A total of 24 rats weighing 200-250 g were divided into four groups, including: control group (C group), ADR injection group (ADR group), SEV pretreatment group (ADR + SEV group) and inhibitor group (ADR + SEV + LY group). H9c2 cells were cultured in vitro and were divided into control group (C group), ADR treatment group (ADR group), and SEV pretreatment group (ADR + SEV group) and inhibitor group (ADR + SEV + LY group) as well. Next, the content of aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) in the serum was detected via Enzyme-Linked Immunosorbent Assay (ELISA). Hematoxylin-eosin (HE) staining assay was performed to determine the severity of myocardial injury. Meanwhile, the apoptosis rate of cells was detected through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay. Additionally, Western blotting (WB) was employed to measure the protein expression levels of phosphorylated (p)-GSK-3β, p-PI3K, Akt and p-Akt. Compared with C group, ADR significantly increased the content of AST, LDH and CK in the serum (p<0.01), reduced protein expression levels of p-GSK-3β, p-PI3K and p-Akt (p<0.01), increased apoptosis rate (p<0.01), and induced myocardial injury. SEV pretreatment significantly alleviated the effect of ADR, manifested as significantly lowered content of AST, LDH and CK in the serum (p<0.01), distinctly elevated protein expression levels of p-GSK-3β, p-PI3K and p-Akt (p<0.01), notably reduced apoptosis rate (p<0.01), and relieved myocardial injury. LY294002 remarkably inhibited the protective effect of SEV against myocardial injury (p<0.01) CONCLUSIONS: SEV is able to prominently ameliorate ADR-induced myocardial injury by regulating the phosphorylation level of the PI3K/Akt/GSK-3β signaling pathway.

Propofol relieves inflammation in MIRI rats by inhibiting Rho/Rock signaling pathway.

The aim of this study was to investigate the effects of propofol on myocardial ischemia-reperfusion injury (MIRI) and its mechanism by establishing in vivo rat models. Sprague-Dawley rats were selected for the construction of MIRI models in vivo. All rats were divided into three groups, including sham operation group (Sham operation), MIRI group and MIRI + propofol group. At 2 h after reperfusion, myocardial tissues and blood samples were collected from rats. The expression levels of serum lactic dehydrogenase (LDH) and creatine kinase-MB (CK-MB), as well as serum interleukin-6 (IL-6), IL-10 and tumor necrosis factor-α (TNF-α), were measured in each group of rats, respectively. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was employed to detect the apoptosis of myocardial cells. Additionally, the messenger ribonucleic acid (mRNA) and protein expressions of Ras homolog gene family, member A (RhoA) and Rho-associated coiled-coil-containing protein kinase 2 (Rock2) were determined via quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. (1) The expression levels of serum LDH and CK-MB were significantly lower in MIRI + propofol group than those in MIRI group (p<0.05). (2) In comparison with MIRI group, MIRI + propofol group exhibited significantly reduced serum IL-6 and TNF-α levels (p<0.01) and elevated serum IL-10 level (p<0.01). (3) Compared with MIRI group, the apoptosis of myocardial cells was remarkably reduced in MIRI + propofol group after IRI (p<0.05). (4) The mRNA and protein expressions of RhoA and Rock2 were significantly lower in MIRI + propofol group than those in MIRI group (p<0.05). Propofol relieves MIRI and inflammation, reduces the level of oxidative stress and represses I/R-induced myocardial cell apoptosis in MIRI rats by inhibiting the activity of the Rho/Rock signaling pathway.

Downregulation of miR-34a ameliorates inflammatory response and apoptosis induced by renal ischemia-reperfusion by promoting Kruppel-like factor 4 expression.

To explore the influence of micro ribonucleic acid (miR)-34a on renal ischemia-reperfusion by regulating Kruppel-like factor 4 (KLF4). A total of 36 Sprague-Dawley (SD) rats weighing 180-200 g were randomly divided into sham operation group (n=12), model group (n=12), and miR-34a inhibitor group (n=12). Renal ischemia-reperfusion modeling was performed in rats of model group and miR-34a inhibitor group. Those in the sham operation group received the same procedures without ligation. 200 μL of miR-34a inhibitor was pre-injected before modeling in rats of miR-34a inhibitor group. An automatic biochemical analyzer was used to detect serum creatinine and urea nitrogen levels in each group of rats, thus reflecting renal functions. The expressions of B-cell lymphoma 2 (Bcl-2), an apoptotic protein, and KLF4, a transcription factor, were detected via Western blotting. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay was conducted to measure the expression levels of miR-34a and KLF4 in rat renal tissues in each group. Immunohistochemistry was employed to determine the expressions of inflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10). Additionally, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was utilized to determine the apoptotic rate in rat kidney tissues in each group. Compared with those in the sham operation group, serum creatinine level, urea nitrogen level, expressions of miR-34a, TNF-α and Bcl-2 (p<0.05) increased, while the levels of KLF4 and IL-10 (p<0.05) decreased in the model group. Apoptosis rate was also higher in the model group than the controls. In comparison with the model group, miR-34a inhibitor group had lowered serum creatinine level, urea nitrogen level, expressions of miR-34a, TNF-α and Bcl-2, and apoptotic rate (p<0.05), but raised levels of KLF4 and IL-10 (p<0.05), showing statistically significant differences. Downregulation of miR-34a ameliorates inflammatory response and apoptosis induced by renal ischemia-reperfusion by promoting KLF4 level, thus improving renal functions in rats.

Could high DNA stainability (HDS) be a valuable indicator of sperm nuclear integrity?

BackgroundThe Sperm Chromatin Structure Assay (SCSA®), in addition to identifying the DNA Fragmentation Index (DFI) also identifies High DNA satiability (HDS), supposed to reflect the nuclear compaction of spermatozoa. However, data on what exactly this parameter reveals, its relevance and usefulness are contradictory. In order to shed light on this situation, spermatozoa of a cohort (N = 397) of infertile men were subjected to the SCSA®, TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling) and CMA3 (Chromomycin A3) tests. In a smaller subcohort (N = 100), aniline blue (AB) and toluidine blue (TB) staining were performed in addition. The objective of this study was thus to answer the question of whether HDS is a relevant and reliable parameter to be taken into account?ResultsHDS does not appear to be a reliable indicator of nuclear immaturity because it shows a weak correlation with the CMA3, AB and TB stains. The low correlation of HDS with sperm DNA fragmentation (TUNEL and SCSA®) and DNA condensation (CMA3, AB and TB) tests suggests that these two parameters could be decoupled. Unlike DFI and TUNEL, HDS has not been shown to correlate with classic clinical situations of male infertility (asthenozoospermia, teratozoospermia or astheno-teratozoospermia).ConclusionHDS correlates poorly with most tests that focus specifically on the level of maturity of the sperm nucleus. To our knowledge, this study is the first to compare SCSA®, TUNEL, AB, TB and CMA3 assays on identical samples. It shows the potency, consistency and limitations of each test and the care that must be taken in their interpretation.

ANO1 relieves pressure overload-induced myocardial fibrosis in mice by inhibiting TGF-β/Smad3 signaling pathway.

The aim of this study was to measure the expression of anoctamin 1 (ANO1) in myocardial tissues of mice with pressure overload-induced myocardial fibrosis, and to further investigate the effect of ANO1 on myocardial fibrosis in mice and its mechanism. A total of 40 male C57/B6 mice aged 6-8 weeks old were divided into 2 groups using a random number table, namely sham operation group (Sham group, n=20) and thoracic aortic constriction group (TAC group, n=20). Meanwhile, 20 ANO1 transgenic (TG) mice aged 6-8 weeks old were enrolled for TAC as TAC + ANO1 TG group. At 8 weeks after TAC, ejection fraction (EF%) and fraction shortening (FS%) in each group of mice were detected via echocardiography. Western blotting and immunofluorescence staining assays were conducted to measure the protein expression of ANO1 in myocardial tissues of mice in each group. The pathological changes in myocardial tissues of mice were evaluated through hematoxylin-eosin (H&E) staining. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay was performed to measure the messenger ribonucleic acid (mRNA) expression levels of hypertrophy markers atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in myocardial tissues of mice in each group. The deposition of collagen fibers in heart tissues was determined by Masson staining assay. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect the apoptosis of myocardial cells and fibroblasts in heart tissues. Additionally, the protein expressions of oxidative stress markers superoxide dismutase 1 (SOD1) and 4-hydroxynonenal (4-HNE) in myocardial tissues were detected as well. Finally, Western blotting was employed to detect the effect of ANO1 overexpression on the expression of transforming growth factor-β (TGF-β)/Smad3 signaling pathway-related proteins in myocardial tissues of mice. At 8 weeks after TAC, ANO1 expression was overtly reduced in myocardial tissues of mice (p<0.05). Echocardiographic results showed that ANO1 overexpression significantly alleviated TAC-induced cardiac function deterioration in mice (p<0.05). The mRNA expression levels of ANP and BNP in myocardial tissues of TAC + ANO1 TG group were evidently lower than those in TAC group (p<0.05). Meanwhile, myocardial interstitial collagen deposition was significantly ameliorated in TAC + ANO1 TG group compared with TAC group (p<0.05). ANO1 overexpression notably mitigated the apoptosis of myocardial cells and oxidative stress in mice with cardiac pressure overload (p<0.05). Western blotting results further indicated that after overexpression of ANO1, the protein levels of TGF-β and phosphorylated Smad3 (p-Smad3) were significantly inhibited in mice undergoing TAC (p<0.05). In the case of cardiac pressure overload in mice, ANO1 is lowly expressed in myocardial tissues. Meanwhile, its overexpression is able to attenuate pressure overload-induced myocardial fibrosis in mice by repressing the TGF-β/smad3 signaling pathway. All our findings indicate that ANO1 can serve as a potential gene target for the treatment of myocardial fibrosis in the future.

Ulinastatin improves myocardial ischemia-reperfusion injury in rats through endoplasmic reticulum stress-induced apoptosis pathway.

To investigate the protective role of ulinastatin (UTI) on myocardial ischemia-reperfusion (I/R) injury in rats via endoplasmic reticulum stress (ERS)-induced apoptosis pathway. A total of 60 rats were randomly divided into normal group (n=20), myocardial I/R model group (model group, n=20), and myocardial I/R model+UTI treatment group (treatment group, n=20). The myocardial function indicators [creatinine (Scr) and creatine kinase (CK)] were detected. Enzyme-linked immunosorbent assay (ELISA) was performed to measure serum levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and matrix metalloproteinase-9 (MMP-9). Meanwhile, the contents of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) in rat left ventricular tissues were determined by ELISA as well. The cardiac function indexes were determined via magnetic resonance imaging (MRI) and echocardiography (ECG). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect the apoptosis of myocardial tissues. Additionally, the expression levels of endoplasmic reticulum stress and apoptosis genes were measured through quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay and Western blotting analysis, respectively. Serum levels of alanine aminotransferase (ALT), CK, and Scr in model group were significantly higher than those in normal group (p<0.05). Besides, rats in model group had significantly lowered SOD, ejection fraction (EF, %), and fractional shortening (FS, %) than those in normal group (p<0.05). In addition, remarkably increased contents of TNF-α, IL-6, MMP-9, MDA, and ROS, as well as higher left ventricular end-diastolic diameter (LVEDd) and left ventricular end-systolic diameter (LVESd) were observed in model group in comparison with normal group (p<0.05). TUNEL staining results revealed that there were more apoptotic cells in model group than that in the other two groups (p<0.05). Expression levels of cysteine aspartic acid-specific protease 12 (Caspase-12) and glucose-regulated protein 78 (GRP78) were evidently higher in model group than those in normal group (p<0.05), while the expression level of B-cell lymphoma 2 (Bcl-2) was clearly lower in model group than that in normal group (p<0.05). UTI treatment partially reversed the above expression changes (p<0.05). UTI has a protective effect against myocardial I/R injury in rats by repressing the occurrence of ERS-induced apoptosis.

Relationship between miR-375 regulating Ndrg2/IL-6/STAT3 signaling pathway and diabetic retinopathy in rats.

To explore the relationship between micro ribonucleic acid (miR)-375 in regulating the N-Myc downstream-regulated gene 2 (Ndrg2)/interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway and diabetic retinopathy (DR) in rats. Thirty Sprague- Dawley rats were randomly divided into Control group (n=10), Model group (n=10), and miR-375 inhibitor group [miR-375 small interfering RNA (siRNA) group, n=10]. The rats in Model group were injected with streptozotocin (STZ) via the tail vein to prepare into rat models of diabetes. The body weight, fasting blood glucose, and retinal barrier permeability of rats in each group were detected. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in rat serum were measured using kits. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of optic ganglion cells in rat retinal tissues. Additionally, the messenger RNA (mRNA) and protein levels of Ndrg2, IL-6 and STAT3 in rat retinal tissues were detected via reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Compared with Control group, Model group had reduced body weight of rats, increased blood glucose and retinal permeability of rats, raised serum MDA content, decreased SOD activity, up-regulated apoptotic rate of optic ganglia, and notably elevated mRNA and protein levels of Ndrg2, IL-6 and STAT3 in retinal tissues. Compared with those in Model group, the body weight of rats declined, the blood glucose of rats rose, the retinal permeability of rats was decreased significantly, the serum MDA content was reduced, the SOD activity was raised, the apoptotic rate of optic ganglia was decreased, and the mRNA and protein levels of Ndrg2, IL-6 and STAT3 in retinal tissues were also decreased significantly in miR-375 siRNA group. MiR-375 inhibitors are able to reduce blood glucose, retinal permeability, and optic ganglion apoptosis in rats with DR, and the mechanism of action may be related to the regulation on the Ndrg2/IL-6/STAT3 signaling pathway.

Effect of miR-133 on apoptosis of trophoblasts in human placenta tissues via Rho/ROCK signaling pathway.

The aim of this study was to explore the role of micro ribonucleic acid (miR)-133 in the apoptosis of human placental trophoblasts through the Ras homolog gene family (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway. The plasma samples were collected from 30 patients with pre-eclampsia (PE) undergoing treatment and 30 healthy subjects (control group) who received physical examination in our hospital. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was utilized to measure the expression of miR-133 in PE patients and healthy people. Meanwhile, blood pressure, urine protein content, liver function, and kidney function were detected in patients of both groups as well. Subsequently, the placental trophoblasts were extracted and transfected with inhibitors and miRNA mimics to suppress and overexpress miR-133, respectively. The transfection efficiency was determined by RT-PCR. The levels of interleukin-6 (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-α) were measured in both groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of trophoblasts. Next, the RT-PCR and Western blotting were carried out to detect the expressions of the Rho/ROCK pathway. Furthermore, the influence of miR-133 on the apoptosis of trophoblasts in human placenta tissues through Rho/ROCK was comprehensively observed. In vivo experiments demonstrated that the urinary protein content, miR-133 level, systolic blood pressure, diastolic blood pressure, and liver function and renal function indexes were significantly elevated in pre-eclampsia (PE) patients in comparison with normal subjects (p<0.05). After transfection of mimics and inhibitors, the expression of miR-133 was remarkably up- and down-regulated, respectively. The content of the inflammatory factors in miR-133 mimics group was overtly higher than the other two groups. The TUNEL staining results showed that the number of apoptotic cells significantly increased and decreased in the miR-133 mimics group and miR-133 inhibitors group, respectively. Subsequent experiments indicated that the expressions of apoptosis gene Caspase3, pathway gene, and protein ROCKI were notably up-regulated in miR-133 mimics group. However, they were evidently down-regulated in miR-133 inhibitors group than in the control group. In addition, a consistent trend was observed in the protein expression level. MiR-133 participates in the development and progression of PE through the Rho/ROCK signaling pathway, which may affect the apoptosis of trophoblasts in the placenta tissues.

Morphine alleviates myocardial ischemia/reperfusion injury in rats by inhibiting TLR4/NF-κB signaling pathway.

The aim of this study was to investigate the effect of morphine on myocardial ischemia/reperfusion (I/R) injury in rats and its underlying mechanism, thereby providing a reference for the prevention and treatment of myocardial I/R injury in clinical practice. A total of 60 male Sprague-Dawley (SD) rats were randomly divided into 3 groups, including: Sham group (n=20), I/R group (n=20) and I/R + morphine group (n=20) using a random number table. The left anterior descending coronary artery (LAD) of rat was ligated and re-canalized, and the I/R model was established in rats. Rats in I/R + sevoflurane (SEV) group were pretreated with 2.5% SEV. Infarction area of heart in each group was detected using triphenyltetrazolium chloride (TTC) test. Ejection fraction % (EF%) and fraction shortening % (FS%) were determined by echocardiography. Hematoxylin-eosin (HE) staining assay was performed to detect the morphological changes of cardiac myocardial cells in each group. Meanwhile, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was adopted to detect the apoptosis of myocardial cells and fibroblasts. In addition, the expression levels of toll-like receptor 4 (TLR4) and p65 in heart samples of rats in each group were measured via immuno-histochemical staining. Finally, the influence of morphine on TLR4/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway was detected using Western blotting. Morphine significantly alleviated I/R-induced cardiac dysfunction in rats, whereas significantly increased EF% and FS% (p<0.05). In addition, morphine evidently inhibited myocardial infarction caused by I/R injury. Meanwhile, it reduced the infarction area from [(59.61±3.41) %] to [(26.67±3.62) %] (p<0.05). The results of HE staining showed that compared with I/R group, I/R + morphine group exhibited remarkably tidier cardiac myofilament, less degradation and necrosis, as well as significantly relieved cellular edema. Immuno-histochemical staining results revealed that morphine overtly reversed decreased expressions of TLR4 and p65 induced by I/R in rats (p<0.05). Furthermore, Western blotting found that morphine significantly inhibited the protein expressions of TLR4 and phosphorylated p65. Morphine clearly alleviates I/R-induced myocardial injury in rats. The possible mechanism may be associated with the inhibition on TLR4/NF-κB signaling pathway.

Melatonin inhibits the inflammation and apoptosis in rats with diabetic retinopathy via MAPK pathway.

To investigate the effect of melatonin on diabetic retinopathy rats through the mitogen-activated protein kinase (MAPK) pathway. A total of 48 Sprague Dawley (SD) rats were randomly divided into normal group (n=12), model group (n=12), melatonin group (n=12), and inhibitor group (n=12). The rats in normal group received no treatment. Those in model group, melatonin group, and inhibitor group were prepared into models of diabetic retinopathy and intraperitoneally injected with normal saline, melatonin, and SB 203580, respectively. After 7 days of intervention, the materials were taken. The expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected through immunohistochemistry. Western blotting was employed to determine the protein expression levels of p38 MAPK, phosphorylated (p)-p38 MAPK, and cysteinyl aspartate specific proteinase-3 (Caspase-3). The messenger ribonucleic acid (mRNA) expression levels of Bax and Bcl-2 were measured via quantitative Polymerase Chain Reaction (qPCR). Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of serum interleukin-1β (IL-1β) and IL-18. The apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL). Based on immunohistochemistry, model group, melatonin group, and inhibitor group exhibited significantly increased positive expression of Bax but notably decreased positive expression of Bcl-2 in comparison with normal group (p<0.05). Compared with those in model group, the positive expression of Bax was clearly reduced, while the positive expression of Bcl-2 was overtly raised in melatonin group and inhibitor group (p<0.05). The results of Western blotting showed that there was no difference in the protein expression of p38 MAPK among all groups (p>0.05). Compared with normal group, the other three groups had remarkably elevated protein expressions of p-p38 MAPK and Caspase-3 (p<0.05). The protein expressions of p-p38 MAPK and Caspase-3 in melatonin group and inhibitor group were significantly lower than those in model group decreased (p<0.05). QPCR assay revealed that the mRNA expression of Bax was markedly lower in normal group than that in the other three groups, while the mRNA expression of Bcl-2 was significantly higher in normal group than that in the other three groups (p<0.05). Compared with model group, melatonin group, and inhibitory group showed clearly declined mRNA expression level of Bax and notably increased mRNA expression level of Bcl-2 (p<0.05). TUNEL results revealed that the apoptosis rate was remarkably elevated in the other three groups compared with that in normal group (p<0.05). In comparison with model group, melatonin group and inhibitor group exhibited significantly reduced apoptosis rate (p<0.05). Melatonin inhibits the inflammation and apoptosis in rats with diabetic retinopathy by repressing the MAPK pathway.

Effects of dexmedetomidine on sepsis-induced liver injury in rats.

To explore the effect of dexmedetomidine (DEX) on sepsis-induced liver injury in rats and the mechanism of action, providing certain references for the prevention and treatment of sepsis-induced liver injury in clinical practice. A total of 60 male Sprague Dawley (SD) rats were randomly divided into 3 groups, namely sham operation group (Sham group, n=20), sepsis-induced liver injury group [lipopolysaccharides (LPS) group, n=20], and sepsis-induced liver injury + DEX group (LPS + DEX group, n=20) using a random number table. Rat models of sepsis-induced liver injury were established by intraperitoneal injection of LPS (10 mg/kg), and at the same time, DEX was intragastrically injected at a dose of 50 μg/kg. After 24 h, the survival analysis curves of each group of rats were plotted. Meanwhile, the levels of liver function indexes and oxidative stress markers were measured at 12 h in each group of rats. Hematoxylin-eosin (H&E) staining assay was carried out to detect the morphological changes of rat liver cells in each group. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining assay was performed to detect the apoptosis level in rat liver tissues in each group. In addition, the expression level of Caspase 3 in three groups of rats was measured through immunohistochemical staining assay. Lastly, the effect of DEX on the protein expression of extracellular signal-regulated kinases 1/2 (ERK1/2) in liver tissues was detected via Western blotting. DEX significantly improved liver dysfunction induced by LPS and raised the 24 h-survival rates of rats (p<0.05). Besides, H&E staining results showed that DEX clearly relieved the pathological damage of rat liver cells caused by LPS. In comparison with LPS group, LPS + DEX group displayed more neatly arranged liver cells, less degradation and necrosis, and evidently attenuated cellular edema. Immunohistochemistry results revealed that DEX significantly reversed the increase in Caspase 3 expression resulting from LPS. The results of the TUNEL staining assay showed that DEX clearly inhibited the apoptosis of rat liver cells induced by LPS. The results of Western blotting revealed that DEX notably reversed the decrease of phosphorylated ERK1/2 (p-ERK1/2) in rat liver tissues compared with LPS group. DEX is able to markedly relieve LPS-induced liver injury in rats and the underlying mechanism may be related to the activation of the ERK1/2 signaling pathway.

A study of regulatory effects of TLR4 and NF-κB on primary biliary cholangitis.

To investigate the regulatory effects of the Toll-like receptor 4 (TLR4) and the nuclear factor kappa-light-chain-enhancer of the activated B cells (NF-κB) on primary biliary cholangitis (PBC) and to analyze the possible mechanisms. A total of 24 C57BL/6 mice were randomly divided into M group (n=12, intraperitoneally injected with polyinosinic acid-polycytidine acid (PolyI:C) for 12 consecutive weeks, 2 times/week) and C group (n=12, intraperitoneally injected with the same volume of normal saline). After 12 weeks, the mice were sacrificed to collect liver tissues. Then, an enzyme-linked immunosorbent assay (ELISA) kit was used to detect the content of interleukin-6 (IL-6), IL-10, and tumor necrosis factor-alpha (TNF-α) in liver tissues. Hematoxylin-eosin (HE) staining assay was performed to observe the pathological changes of liver tissues, and measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in peripheral blood of mice. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) staining was applied to determine cell apoptosis in liver tissues. The relative messenger ribonucleic acid (mRNA) expression levels of TLR4 and NF-κB in liver tissues were detected by quantitative Polymerase Chain Reaction (qPCR). Western blotting was adopted to measure the protein expressions of TLR4, NF-κB, myeloid differentiation factor 88 (MyD88), B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax), and Caspase-3. Compared with that in C group, the content of IL-6 and TNF-α in liver tissues in M group was significantly increased (p<0.01), but the level of IL-10 was statistically downregulated (p<0.01). According to HE staining, liver damage of mice in M group was evidently severer than that in C group, and the levels of ALT and AST in M group were significantly higher than those in C group (p<0.01). The amount of TUNEL-positive cells in liver tissues in M group was significantly greater than that in C group (p<0.01). The levels of TLR4 and NF-κB mRNA in liver tissues from M group were significantly elevated in comparison with the C group (p<0.01). Compared with those in C group, the expressions of TLR4, NF-κB, MyD88, and Caspase-3 proteins in M group showed statistical increases in liver tissues (p<0.01), whereas that of Bcl-2/Bax was significantly declined (p<0.01). PBC activates the TLR4/MyD88/NF-κB signaling pathway, induces the release of inflammatory factors and produces a large number of apoptotic proteins, which results in liver damage and cell apoptosis in mice.

TGF-β and HSP70 profiles during transformation of yak hair follicles from the anagen to catagen stage.

Transforming growth factor-β (TGF-β) and heat shock protein 70 (HSP70) are important for the hair follicle (HF) cycle, but it is unclear whether they participate in HF regression in yak skin. In this study, we investigated the role of TGF-β, TGF-βRII, and HSP70 in the transition from anagen to catagen of HFs. The results showed that TGF-β2 transcription was significantly higher than that of TGF-β1 and TGF-β3 in the same periods. Meanwhile, the expressions of TGF-β2, TGF-βRII, and caspase-3 were higher in the catagen phase than that in mid-anagen, and some TGF-βRII-positive HF cells were terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-positive. Moreover, the HSP70 protein levels in mid-anagen were higher than those in late-anagen and catagen. These results suggested that TGF-β2 plays a major role in catagen induction in yak HFs, which might be achieved via TGF-βRII-mediated apoptosis in HF epithelial cells. In contrast, HSP70 might protect epithelial cells from apoptosis and ultimately inhibit HF regression. In conclusion, TGF-β2 has positive effects, whereas HSP70 has negative effects, on catagen induction.

Effect of miR-21/TLR4/NF-κB pathway on myocardial apoptosis in rats with myocardial ischemia-reperfusion.

The aim of the study was to investigate the influences of micro ribonucleic acid (miR)-21 and downstream Toll-like receptor 4 (TLR4)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway on myocardial apoptosis induced by myocardial ischemia-reperfusion (I/R) injury in rats. Recombinant adeno-associated virus rAAV9-ZsGreen-pre-miR-21 and blank control virus were constructed. A total of 48 Sprague-Dawley (SD) rats were randomly divided into S1 group (open chest only), S2 group (transfection with blank virus + open chest), I/R1 group (transfection with blank virus + 6 d of myocardial I/R), and I/R2 group (transfection with miR-21 + 6 d of myocardial I/R). The cardiac function and myocardial infarct size of rats were evaluated in each group. Quantitative Polymerase Chain Reaction (qPCR) was applied to measure the expression level of miR-21 in the myocardium. The level of myocardial apoptosis in each group was detected through terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. Western blotting was performed to determine the protein expression levels of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax), Caspase-3, TLR4, and NF-κB in the myocardium. The content of interleukin-6 (IL-6) and IL-10 was measured using enzyme-linked immunosorbent assay (ELISA) kits. The cardiac function of rats in I/R1 and I/R2 groups was significantly lower than that in S1 and S2 groups (p<0.01). Rats in I/R2 group had better cardiac function than those in I/R1 group (p<0.01). In I/R1 group, the level of myocardial apoptosis of rats was overtly increased compared with that in S1, S2, and I/R2 groups (p<0.01), while the expression level of miR-21 in myocardium was evidently lower than that in S1, S2, and I/R2 groups (p<0.01). Compared with S1, S2, and I/R2 groups, I/R1 group had markedly decreased Bcl-2/Bax expression level and IL-10 content and overtly elevated expression levels of Caspase-3, p-TLR4, p-NF-κB, and IL-6 content in the myocardium (p<0.01). Myocardial I/R injury in rats leads to decreased expression of miR-21. The overexpression of miR-21 is able to effectively inhibit the TLR4/NF-κB pathway and reduce the level of myocardial apoptosis of rats and the release of inflammatory factors.

Synergistic effect of rapamycin and metformin against germ cell apoptosis and oxidative stress after testicular torsion/detorsion-induced ischemia/reperfusion in rats.

The aim of this study was to investigate the effects of rapamycin (rapa) and metformin (met), combined administration on testicular torsion-detorsion (T/D) injury. A total of 108 male rats were divided randomly into six groups (n = 18), control, sham-operated, T/D, T/D + met (100 mg/kg), T/D + rapa (0.25 mg/kg) and T/D + met (100 mg/kg)+rapa (0.25 mg/kg). Except for the control and sham groups, torsion was created by rotating the right testis 720° in a clockwise direction for 1 h. Treatment groups received drug intraperitoneally, 30 min before detorsion. The right testis of 6 animals from each group was excised 4 h after detorsion for the measurement of lipid peroxidation, caspase-3, and antioxidant enzyme activities. Histopathological changes and germ cell apoptosis were determined by measuring mean of seminiferous tubules diameters (MSTD) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) test in rest of animals, 24 h after detorsion. In T/D group tissue malondialdehyde (MDA) level and caspase-3 activity increased and the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) decreased in comparison with the control group after detorsion. Met and rapa separately pre-treatment reduced MDA and caspase-3 levels, normalized antioxidant enzyme activities, reduced germ cell apoptosis and improved the MSTD in comparison with T/D group. However combined administration of met and rapa indicated a significant augmented effect as compared to the individual drug interventions on the reversal of T/D induced oxidative stress, apoptosis, and histologic changes, suggesting a synergistic response. Thus, this study shows that rapa and met combination have significant synergistic effects against oxidative stress and apoptosis and opens up further possibilities for the design of new combinatorial therapies to prevent tissue damage after ischemia-reperfusion (I/R).

Correlations of neuronal apoptosis with expressions of c-Fos and c-Jun in rats with post-ischemic reconditioning damage.

Transcription factors (c-Fos and c-Jun) have been considered to play roles in the initiation of programmed nerve cell death. However, the roles of c-Fos and c-Jun protein expressions in neuronal apoptosis of rats with post-ischemic reconditioning damage were not clarified. Therefore, the aim of this study was to investigate the correlations of protein expressions of c-Fos and c-Jun with neuronal apoptosis of rats with post-ischemic reconditioning damage. Rat models of post-ischemic reconditioning were established firstly. Then, apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, and the gene expression levels of apoptosis-related proteins [cytochrome c (Cyt c), B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax)] were detected by reverse transcription-polymerase chain reaction (RT-PCR). Lastly, Western blotting was used to determine the protein expression levels of c-Fos and c-Jun, and the expressions of c-Fos and c-Jun in brain tissues of models were measured by immunohistochemistry. Treatment group had significantly increased malonaldehyde (MDA) level and significantly decreased superoxide dismutase (SOD) activity in rat cortex compared with those in control group (p<0.05). The number of TUNEL positive cells in the right cortex of rats in the treatment group was clearly higher than that in control group. Among them, post-ischemic reperfusion group had reduced level of Bax in the cytoplasm, but increased Bax level in the mitochondrion, and lowered expression level of Bcl-2 in both mitochondrion and cytoplasm in comparison with control group. Dynamic detection results of c-Jun were in synchronization with those of apoptosis proteins, and maximum expression occurred at 24 h after treatment. c-Jun may play a role in the initiation of apoptotic cell death in these neurons.

Targeting intestinal epithelial cell–programmed necrosis alleviates tissue injury after intestinal ischemia/reperfusion in rats

BackgroundIntestinal dysfunction, especially acute pathologies linked to intestinal ischemia/reperfusion (I/R) injury, is profoundly affected by inflammation and improper execution of cell death. Few studies have examined the efficacy of combined strategies in regulated intestinal epithelial necrosis after intestinal I/R. Here, we evaluated the functional interaction between poly (adenosine diphosphate-ribose) polymerase 1 (PARP-1)-induced parthanatos and receptor-interacting protein 1/3 (RIP1/3) kinase-induced necroptosis in the pathophysiological course of acute ischemic intestinal injury. MethodsAnesthetized adult male Sprague–Dawley rats were subjected to superior mesenteric artery occlusion consisting of 1.5 h of ischemia and 6 h of reperfusion. The PARP-1-specific inhibitor PJ34 (10 mg/kg) and the RIP1-specific inhibitor Necrostatin-1 (1 mg/kg) were intraperitoneally administered 30 min before the induction of ischemia. ResultsIntestinal I/R was found to result in PARP-1 activation and RIP1/3-mediated necrosome formation. PJ34 or Necrostatin-1 treatment significantly improved the mucosal injury, while the combined inhibition of PARP-1 and RIP1/3 conferred optimal protection of the intestine. Meanwhile, results from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay showed a decrease in intestinal epithelial cell death. Interestingly, we further showed that PARP-1 might act as a downstream signaling molecule of RIP1 in the process of I/R-induced intestinal injury and that the RIP1/PARP-1-dependent cell death signaling pathway functioned independently of caspase 3 inhibition. ConclusionsThe results of our study provide a molecular basis for combination therapy that targets both pathways of regulated necrosis (parthanatos and necroptosis), to treat acute intestinal I/R-induced intestinal epithelial barrier disruption.