Distinct Roles of DRP1 in Conventional and Alternative Mitophagy in Obesity Cardiomyopathy.

BACKGROUND: Obesity induces cardiomyopathy characterized by hypertrophy and diastolic dysfunction. Whereas mitophagy mediated through an Atg7 (autophagy related 7)-dependent mechanism serves as an essential mechanism to maintain mitochondrial quality during the initial development of obesity cardiomyopathy, Rab9 (Ras-related protein Rab-9A)-dependent alternative mitophagy takes over the role during the chronic phase. Although it has been postulated that DRP1 (dynamin-related protein 1)-mediated mitochondrial fission and consequent separation of the damaged portions of mitochondria are essential for mitophagy, the involvement of DRP1 in mitophagy remains controversial. We investigated whether endogenous DRP1 is essential in mediating the 2 forms of mitophagy during high-fat diet (HFD)-induced obesity cardiomyopathy and, if so, what the underlying mechanisms are. METHODS: Mice were fed either a normal diet or an HFD (60 kcal %fat). Mitophagy was evaluated using cardiac-specific Mito-Keima mice. The role of DRP1 was evaluated using tamoxifen-inducible cardiac-specific Drp1knockout (Drp1 MCM) mice. RESULTS: Mitophagy was increased after 3 weeks of HFD consumption. The induction of mitophagy by HFD consumption was completely abolished in Drp1 MCM mouse hearts, in which both diastolic and systolic dysfunction were exacerbated. The increase in LC3 (microtubule-associated protein 1 light chain 3)-dependent general autophagy and colocalization between LC3 and mitochondrial proteins was abolished in Drp1 MCM mice. Activation of alternative mitophagy was also completely abolished in Drp1 MCM mice during the chronic phase of HFD consumption. DRP1 was phosphorylated at Ser616, localized at the mitochondria-associated membranes, and associated with Rab9 and Fis1 (fission protein 1) only during the chronic, but not acute, phase of HFD consumption. CONCLUSIONS: DRP1 is an essential factor in mitochondrial quality control during obesity cardiomyopathy that controls multiple forms of mitophagy. Although DRP1 regulates conventional mitophagy through a mitochondria-associated membrane-independent mechanism during the acute phase, it acts as a component of the mitophagy machinery at the mitochondria-associated membranes in alternative mitophagy during the chronic phase of HFD consumption.

Characterization of a WD-repeat family protein WDR3 in the brain of WDR3 hetero knockout mice.

The nuclear protein WDR3 is a member of the WD-repeat family and is a component of the 18S pre-rRNA processing complex. However, the expression and function of WDR3 in the brain remains unknown. To characterize WDR3 in the adult mouse brain, we developed Wdr3 heterozygous knockout (WDR3-HKO) mice. Notably, no homozygous Wdr3 knockout mice were born, suggesting that complete absence of WDR3 causes lethal abnormalities during embryogenesis. Brain Wdr3 mRNA expression was significantly reduced to 60% in the WDR3-HKO mice compared to wild type (WT) mice, while the expression of 18S rRNA did not decline. Using immunohistochemistry and X-gal staining, we demonstrated that WDR3 is widely expressed in the mouse brain, especially in the hippocampus, habenular nucleus, and cerebellum. We observed no differences in body weight during adulthood or developmental weight gain between the WDR3-HKO and WT mice. Interestingly, WDR3-HKO mice exhibited a slight but significant increase in spontaneous locomotor activity compared to WT littermates. In conclusion, the WDR3-HKO mice showed no significant phenotypic changes. Further studies are required to explore the behavioral characteristics of WDR3-HKO mice.

[Effects of CACNA1H gene knockout on autistic-like behaviors and the morphology of hippocampal neurons in mice].

OBJECTIVE: To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice. METHODS: In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H-/--Thy1+(KO-GFP) and CACNA1H+/+-Thy1+ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed. RESULTS: In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F1, 14=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F1, 14=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05). CONCLUSION: CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.

Osteopontin on the Dental Implant Surface Promotes Direct Osteogenesis in Osseointegration.

After dental implantation, osteopontin (OPN) is deposited on the hydroxyapatite (HA) blasted implant surface followed by direct osteogenesis, which is significantly disturbed in Opn-knockout (KO) mice. However, whether applying OPN on the implant surface promotes direct osteogenesis remains unclarified. This study analyzed the effects of various OPN modified protein/peptides coatings on the healing patterns of the bone-implant interface after immediately placed implantation in the maxilla of four-week-old Opn-KO and wild-type (WT) mice (n = 96). The decalcified samples were processed for immunohistochemistry for OPN and Ki67 and tartrate-resistant acid phosphatase histochemistry. In the WT mice, the proliferative activity in the HA binding peptide-OPN mimic peptide fusion coated group was significantly higher than that in the control group from day 3 to week 1, and the rates of OPN deposition and direct osteogenesis around the implant surface significantly increased in the recombinant-mouse-OPN (rOPN) group compared to the Gly-Arg-Gly-Asp-Ser peptide group in week 2. The rOPN group achieved the same rates of direct osteogenesis and osseointegration as those in the control group in a half period (week 2). None of the implant surfaces could rescue the direct osteogenesis in the healing process in the Opn-KO mice. These results suggest that the rOPN coated implant enhances direct osteogenesis during osseointegration following implantation.

Construction of a Spink5 conditional knockout mouse model and analysis of its phenotype / 中华皮肤科杂志

Objective:To construct a serine protease inhibitor Kazal type-5 (Spink5) conditional knockout mouse model, and to identify its phenotype.Methods:B cell-specific Spink5 conditional knockout mice of genotype Mb1 cre/+Spink5 floxp/floxp were constructed by using clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9) technology, and served as the knockout group. Mice of genotype Mb1 +/+Spink5 floxp/floxp served as the control group. The mice of genotype Mb1 cre/+Spink5 floxp/floxp or Mb1 +/+Spink5 floxp/floxp were sacrificed when they were 4 to 6 weeks old, splenic mononuclear cells were isolated, and B lymphocytes and non-B lymphocytes were sorted by flow cytometry and fluorescence-activated cell sorting. Genotype identification was performed by PCR, and protein expression of lymphoepithelial Kazal-type-related inhibitor (LEKTI) was determined by Western blot analysis. Skin tissues were resected from the mice, and subjected to hematoxylin-eosin staining for measuring the epidermal thickness. Immunofluorescence staining was performed to determine fluorescence intensity of LEKTI protein in the mouse skin tissues. Paired t test or two-independent-sample t test was used for comparisons between groups. Results:Genotype identification results demonstrated that the stable B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed. Western blot analysis revealed that the relative protein expression of LEKTI in the B lymphocytes in the knockout group was 0.01 ± 0.02, which was significantly lower than that in the non-B lymphocytes in the knockout group (0.66 ± 0.11, t = 9.99, P < 0.001) , and that in the B lymphocytes in the control group (1.08 ± 0.13, t = 13.78, P < 0.001) . Among 39 mice in the knockout group, 4 presented with dry skin and scattered scaly hypertrophic maculopapules. The epidermal thickness of the lesional skin tissues in the knockout group was 90.42 ± 21.31 μm, significantly higher than that of the non-lesional skin tissues in the knockout group (29.71 ± 3.63 μm, t = 5.05, P = 0.002) and that of normal skin tissues in the control group (12.42 ± 2.21 μm, t = 6.74, P < 0.001) . Immunofluorescence staining showed no significant difference in the fluorescence intensity of LEKTI protein among the lesional skin tissues (46.21 ± 1.21) , non-lesional skin tissues (46.62 ± 2.13) in the knockout group and normal skin tissues in the control group (47.69 ± 1.71, P > 0.05) . Conclusion:The B lymphocyte-specific Spink5 conditional knockout mouse model was successfully constructed, which provides a basis for further exploring mechanisms underlying skin barrier defects and immune dysfunction in Netherton syndrome.

Effects of CACNA1H gene knockout on autistic-like behaviors and the morphology of hippocampal neurons in mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice.@*METHODS@#In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H-/--Thy1+(KO-GFP) and CACNA1H+/+-Thy1+ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed.@*RESULTS@#In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F1, 14=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F1, 14=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05).@*CONCLUSION@#CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.

Inhibition of Soluble Epoxide Hydrolase Attenuates Bosutinib-Induced Blood Pressure Elevation.

[Figure: see text].

Rapid Way to Generate Mouse Models for In Vivo Studies of the Endothelium.

A single layer of squamous endothelial cells (ECs), the endothelium, regulates the flow of substance and fluid into and out of a tissue. The endothelium is also involved in vasculogenesis, the formation of new blood vessels, which is a crucial process for organ development in the embryo and fetus. Because most murine mutations of genes involved in EC development cause early embryo lethality, EC-specific conditional knockout (cKO) mouse models are indispensable for in vivo studies. cKO mice including the floxed allele can be generated through advanced approaches including embryonic stem cell-mediated gene targeting or the CRISPR/Cas system. EC-specific mouse models can be generated through further breeding of floxed mice with a Cre driver line, the latest information of which is available in the Jackson Cre Repository or the EUCOMMTOOLS project. Because it takes a long time (generally 1-2 years) to generate EC-specific mouse models, researchers must thoroughly design and plan a breeding strategy before full-scale mouse experiments, which saves time and money for in vivo study. In summary, revolutionary technical advances in embryo manipulation and assisted reproduction technologies have made it easier to generate EC-specific mouse models, which have been used as essential resources for in vivo studies of the endothelium.

MondoA Is Required for Normal Myogenesis and Regulation of the Skeletal Muscle Glycogen Content in Mice.

Background: Skeletal muscle is the largest tissue in the human body, and it plays a major role in exerting force and maintaining metabolism homeostasis. The role of muscle transcription factors in the regulation of metabolism is not fully understood. MondoA is a glucose-sensing transcription factor that is highly expressed in skeletal muscle. Previous studies suggest that MondoA can influence systemic metabolism homeostasis. However, the function of MondoA in the skeletal muscle remains unclear. Methods: We generated muscle-specific MondoA knockout (MAKO) mice and analyzed the skeletal muscle morphology and glycogen content. Along with skeletal muscle from MAKO mice, C2C12 myocytes transfected with small interfering RNA against MondoA were also used to investigate the role and potential mechanism of MondoA in the development and glycogen metabolism of skeletal muscle. Results: MAKO caused muscle fiber atrophy, reduced the proportion of type II fibers compared to type I fibers, and increased the muscle glycogen level. MondoA knockdown inhibited myoblast proliferation, migration, and differentiation by inhibiting the phosphatase and tensin homolog (PTEN)/phosphoinositide 3-kinase (PI3K)/Akt pathway. Further mechanistic experiments revealed that the increased muscle glycogen in MAKO mice was caused by thioredoxin-interacting protein (TXNIP) downregulation, which led to upregulation of glucose transporter 4 (GLUT4), potentially increasing glucose uptake. Conclusion: MondoA appears to mediate mouse myofiber development, and MondoA decreases the muscle glycogen level. The findings indicate the potential function of MondoA in skeletal muscle, linking the glucose-related transcription factor to myogenesis and skeletal myofiber glycogen metabolism.

Blowing up Neural Repair for Stroke Recovery: Preclinical and Clinical Trial Considerations.

The repair and recovery of the brain after stroke is a field that is emerging in its preclinical science and clinical trials. However, recent large, multicenter clinical trials have been negative, and conflicting results emerge on biological targets in preclinical studies. The coalescence of negative clinical translation and confusion in preclinical studies raises the suggestion that perhaps the field of stroke recovery faces a fate similar to stroke neuroprotection, with interesting science ultimately proving difficult to translate to the clinic. This review highlights improvements in 4 areas of the stroke neural repair field that should reorient the field toward successful clinical translation: improvements in rodent genetic models of stroke recovery, consideration of the biological target in stroke recovery, stratification in clinical trials, and the use of appropriate clinical trial end points.

Dentin Matrix Protein 1 Compensates for Lack of Osteopontin in Regulating Odontoblastlike Cell Differentiation after Tooth Injury in Mice.

INTRODUCTION: Although dentin matrix protein 1 (DMP1) and osteopontin (OPN) act as substrates and signaling molecules for odontoblastlike cell differentiation after tooth injury, the mutual interaction between these proteins in the mechanism of odontoblastlike cell differentiation remains to be clarified. This study aimed to elucidate the role of DMP1 and OPN in regulating odontoblastlike cell differentiation after tooth injury. METHODS: A groove-shaped cavity was prepared on the mesial surface of the upper first molars in wild-type and Opn knockout (KO) mice. The demineralized paraffin sections were processed for immunohistochemistry for nestin and DMP1 and in situ hybridization for Dmp1. For the in vitro assay, the experiments of organ culture for evaluating dentin-pulp complex regeneration using small interfering RNA treatment were performed. RESULTS: Once preexisting odontoblasts died, nestin-positive newly differentiated odontoblastlike cells were arranged along the pulp-dentin border and began to express DMP1/Dmp1. In Opn KO mice, the expression of DMP1/Dmp1 was up-regulated compared with that of wild-type mice. The in vitro assay showed that the gene suppression of Dmp1 by small interfering RNA showed a tendency to decrease the differentiation rate of odontoblastlike cells from 70.1% to 52.2% in wild-type teeth. In addition, the suppression of Dmp1 in Opn KO teeth tended to lead to the inhibition of odontoblastlike cell differentiation. CONCLUSIONS: These results suggest that the expression of Dmp1 is up-regulated in Opn KO mice both in vivo and in vitro, and DMP1 compensates for the lack of OPN in regulating odontoblastlike cell differentiation after tooth injury.

Diet-Induced Obesity Promotes Kidney Endothelial Stiffening and Fibrosis Dependent on the Endothelial Mineralocorticoid Receptor.

Obesity is characterized by enhanced MR (mineralocorticoid receptor) activation, vascular stiffness, and associated cardiovascular and kidney disease. Consumption of a Western-style diet (WD), high in saturated fat and refined carbohydrates, by female mice, leads to obesity and vascular stiffening. Use of ECMR (endothelial cell-specific MR) knockout mice supports that ECMR activation is critical for development of vascular and cardiac fibrosis and stiffening. However, the role of ECMR activation in kidney inflammation and fibrosis remains unknown. We hypothesized that cell-specific deletion of ECMR would prevent WD-induced central aortic stiffness and protect the kidney from endothelial dysfunction and vascular stiffening. Four-week-old female ECMR KO and wild-type mice were fed either mouse chow or WD for 16 weeks. WD feeding increased body weight and fat mass, proteinuria, as well as vascular stiffness indices (pulse wave velocity and kidney artery stiffening) and impaired endothelial-dependent vasodilatation without blood pressure changes. The WD-induced kidney arterial stiffening was associated with attenuated eNOS (endothelial NO synthase) activation, increased oxidative stress, proinflammatory immune responses, alterations in extracellular matrix degradation pathways, and fibrosis. ECMR deletion prevented these abnormalities by improving eNOS activation and reducing macrophage proinflammatory M1 polarization, expression of TG2 (transglutaminase 2), and MMP (matrix metalloproteinase)-9. Our data support the concept that ECMR activation contributes to endothelial dysfunction, increased kidney artery fibrosis/stiffening, and impaired NOS (NO synthase) activation, processes associated with macrophage infiltration and polarization, inflammation, and oxidative stress, collectively resulting in tubulointerstitial fibrosis in females consuming a WD.

Loss of 2 Akt (Protein Kinase B) Isoforms in Hematopoietic Cells Diminished Monocyte and Macrophage Survival and Reduces Atherosclerosis in Ldl Receptor-Null Mice.

Objective- Macrophages express 3 Akt (protein kinase B) isoforms, Akt1, Akt2, and Akt3, which display isoform-specific functions but may be redundant in terms of Akt survival signaling. We hypothesize that loss of 2 Akt isoforms in macrophages will suppress their ability to survive and modulate the development of atherosclerosis. Approach and Results- To test this hypothesis, we reconstituted male Ldlr-/- mice with double Akt2/Akt3 knockout hematopoietic cells expressing only the Akt1 isoform (Akt1only). There were no differences in body weight and plasma lipid levels between the groups after 8 weeks of the Western diet; however, Akt1only→ Ldlr-/- mice developed smaller (57.6% reduction) atherosclerotic lesions with more apoptotic macrophages than control mice transplanted with WT (wild type) cells. Next, male and female Ldlr-/- mice were reconstituted with double Akt1/Akt2 knockout hematopoietic cells expressing the Akt3 isoform (Akt3only). Female and male Akt3only→ Ldlr-/- recipients had significantly smaller (61% and 41%, respectively) lesions than the control WT→ Ldlr-/- mice. Loss of 2 Akt isoforms in hematopoietic cells resulted in markedly diminished levels of white blood cells, B cells, and monocytes and compromised viability of monocytes and peritoneal macrophages compared with WT cells. In response to lipopolysaccharides, macrophages with a single Akt isoform expressed low levels of inflammatory cytokines; however, Akt1only macrophages were distinct in expressing high levels of antiapoptotic Il10 compared with WT and Akt3only cells. Conclusions- Loss of 2 Akt isoforms in hematopoietic cells, preserving only a single Akt1 or Akt3 isoform, markedly compromises monocyte and macrophage viability and diminishes early atherosclerosis in Ldlr-/- mice.

Effect of tumor necrosis factor-related apoptosis-inducing ligand gene knock-out on the gut microbiota in mice with dextran sodium sulphate induced colitis / 中华消化杂志

Objective@#To investigate the influence of tumor necrosis factor-related apoptosis-inducing ligant (TRAIL) deficiency on mice colitis and the gut microbiota composition by inclding the expermental colitis model in tumor necrosis factor-related apoptosis-inducing ligand gene knockout (TRAIL-/-) mice.@*Methods@#C57BL/6 TRAIL-/- mice and wild type (WT) mice were selected and assigned into TRAIL-/- control group (eight mice), TRAIL-/- colitis group (16 mice), WT control group (eight mice) and WT colitis group (16 mice). The mice of two colitis groups were oral administrated with 3.5% dextran sulphate sodium (DSS) in drinking water for seven consecutive days to induce experimental colitis model. The severity of colitis was evaluated by clinical appearance and histopathological examination. The colonic tissue samples of mice were collected and microbiota profile was analyzed by 16S rDNA sequencing method. USEARCH software and R language were used to analyze the difference of gut microbiota among TRAIL-/- control group, TRAIL-/- colitis group, WT control group and WT colitis group. T test and Mann-Whitney U test were used for statistical analysis.@*Results@#After modeling, the disease activity index (DAI) of WT colitis mice and TRAIL-/- colitis mice both gradually increased over time. Furthermore, compared with colitis mice, TRAIL-/- colitis mice developed body weight loss, diarrhea and hemafecia earlier. On the seventh day after modeling, the percentage of body weight loss of TRAIL-/- colitis mice and WT colitis mice was (28.98±2.84)% and (17.87±3.70)%, respectively; and the difference was statistically significant (t=9.53, P<0.01). The length of colon of TRAIL-/- colitis mice was shorter than that of WT colitis mice ((4.63±0.28) cm vs. (6.02±0.41) cm), and the difference was statistically significant (t=11.20, P<0.01). The DAI of TRAIL-/- colitis mice was higher than that of WT colitis mice (3.00±0.00 vs. 2.32±0.05), and the difference was statistically significant (t=54.40, P<0.01). The histological score of TRAIL-/- colitis mice was higher than that of WT colitis mice (6.19±0.25 vs. 3.87±0.22), and the difference was statistically significant (t=27.87, P<0.01). Under the microscope, colonic mucosal epithelial injury, crypt structure destruction and inflammatory cell infiltration were more obvious in TRAIL-/- colitis mice than in WT colitis mice. The alpha diversity of colonic flora was more significant in TRAIL-/- colitis group compared with that of WT colitis group. At the family level, the relative richness of Deferribacteraceae, Ruminococcaceae, Rikenellaceae, F16 and Paraprevotellaceae significantly increased in TRAIL-/- colitis group, but the relative richness of Enterococcaceae obviously reduced ((19.839±19.991)% vs. (7.224±11.241)%, (3.564±2.543)% vs.(2.861±3.821)%, (0.123±0.066)% vs. (0.068±0.049)%, (0.032±0.033)% vs. (0.006±0.011)%, (0.153±0.098)% vs. (0.062±0.054)% and (0.013±0.027)% vs. (0.054±0.121)%, respectively; U=51, 69, 53, 35, 49 and 69, respectively; P<0.01 and 0.05, respectively). In addition, at the genus level the relative richness of Oscillospira, Mucispirillum and Cytophaga in TRAIL-/- colitis group remarkably elevated, and the relative richness of Enterococcus significantly decreased ((2.363±2.147)% vs. (1.813±2.847)%, (19.839±19.991)% vs. (7.223±11.241)%, (0.104±0.153)% vs. (0.046±0.069)% and (0.076±0.049)% vs. (0.135±0.074)%, respectively; U=70, 51, 66 and 65, respectively; P <0.05 and 0.01, respectively).@*Conclusion@#TRAIL deficiency aggravate DSS-induced colitis, and increase the alpha diversity of colonic microbiota in colitis mice.

Effect of tumor necrosis factor-related apoptosis-inducing ligand gene knock-out on the gut microbiota in mice with dextran sodium sulphate induced colitis / 中华消化杂志

Objective To investigate the influence of tumor necrosis factor-related apoptosis-inducing ligant (TRAIL) deficiency on mice colitis and the gut microbiota composition by inclding the expermental colitis model in tumor necrosis factor-related apoptosis-inducing ligand gene knockout ( TRAIL-/-) mice. Methods C57BL/6 TRAIL-/-mice and wild type (WT) mice were selected and assigned into TRAIL-/-control group (eight mice), TRAIL-/-colitis group (16 mice), WT control group (eight mice) and WT colitis group (16 mice).The mice of two colitis groups were oral administrated with 3.5% dextran sulphate sodium (DSS) in drinking water for seven consecutive days to induce experimental colitis model .The severity of colitis was evaluated by clinical appearance and histopathological examination .The colonic tissue samples of mice were collected and microbiota profile was analyzed by 16S rDNA sequencing method.USEARCH software and R language were used to analyze the difference of gut microbiota among TRAIL-/-control group, TRAIL-/-colitis group, WT control group and WT colitis group .T test and Mann-Whitney U test were used for statistical analysis . Results After modeling, the disease activity index (DAI) of WT colitis mice and TRAIL-/-colitis mice both gradually increased over time .Furthermore, compared with colitis mice, TRAIL-/-colitis mice developed body weight loss, diarrhea and hemafecia earlier .On the seventh day after modeling , the percentage of body weight loss of TRAIL-/-colitis mice and WT colitis mice was (28.98 ±2.84)%and (17.87 ±3.70)%, respectively; and the difference was statistically significant (t＝9.53, P?0.01).The length of colon of TRAIL-/-colitis mice was shorter than that of WT colitis mice ((4.63 ±0.28) cm vs.(6.02 ±0.41) cm), and the difference was statistically significant (t＝11.20, P?0.01).The DAI of TRAIL-/-colitis mice was higher than that of WT colitis mice (3.00 ±0.00 vs.2.32 ±0.05), and the difference was statistically significant (t ＝54.40, P? 0.01).The histological score of TRAIL-/-colitis mice was higher than that of WT colitis mice (6.19 ±0.25 vs. 3.87 ±0.22), and the difference was statistically significant (t ＝27.87, P?0.01).Under the microscope, colonic mucosal epithelial injury , crypt structure destruction and inflammatory cell infiltration were more obvious in TRAIL-/-colitis mice than in WT colitis mice.The alpha diversity of colonic flora was more significant in TRAIL-/-colitis group compared with that of WT colitis group .At the family level, the relative richness of Deferribacteraceae, Ruminococcaceae, Rikenellaceae, F16 and Paraprevotellaceae significantly increased in TRAIL-/-colitis group, but the relative richness of Enterococcaceae obviously reduced ((19.839 ±19.991)%vs. (7.224 ±11.241)%, (3.564 ±2.543)% vs.(2.861 ±3.821)%, (0.123 ±0.066)% vs.(0.068 ± 0.049)%, (0.032 ±0.033)% vs.(0.006 ±0.011)%, (0.153 ±0.098)% vs.(0.062 ±0.054)% and (0.013 ±0.027)%vs.(0.054 ±0.121)%, respectively; U＝51, 69, 53, 35, 49 and 69, respectively; P? 0.01 and 0.05, respectively).In addition, at the genus level the relative richness of Oscillospira, Mucispirillum and Cytophaga in TRAIL-/-colitis group remarkably elevated , and the relative richness of Enterococcus significantly decreased ((2.363 ±2.147)% vs.(1.813 ±2.847)%, (19.839 ±19.991)% vs.(7.223 ± 11.241)%, (0.104 ±0.153)%vs.(0.046 ±0.069)% and (0.076 ±0.049)% vs.(0.135 ±0.074)%, respectively; U＝70, 51, 66 and 65, respectively; P ?0.05 and 0.01, respectively).Conclusion TRAIL deficiency aggravate DSS-induced colitis, and increase the alpha diversity of colonic microbiota in colitis mice .

Study on Role and Mechanism of Claudin-2 in Formation of Cholesterol Gallstone / 胃肠病学

Background: Claudin protein family is an important component for construction of tight cell junction. Claudin-2 is a forming protein of paracellular channel and is highly expressed in hepatocytes and bile duct cells, which has selective permeability of water molecules and cations. Claudin-2 plays a vital role in the physiological regulation of digestive and urinary systems. Aims: To investigate the role and mechanism of claudin-2 in formation of cholesterol gallstone and bile secretion. Methods: Liver and gallbladder tissues from claudin-2 knockout (Cldn-2-/-) and wild type (Cldn-2+/+) mice were collected. Expressions of claudin family protein were determined by Western blotting. HE staining, immunofluorescence staining, electron microscope were used to analyze the effect of claudin-2 gene knockout on liver and gallbladder tissue structure. Regulatory roles of claudin-2 in bile component and flow were analyzed. Results: Claudin family proteins were highly expressed in liver and gallbladder tissue in Cldn-2+/+ mice. No significant difference in structure of liver and gallbladder tissue was found between Cldn-2-/- mice and Cldn-2+/+ mice. Compared with Cldn-2+/+ mice, bile flow rate was significantly decreased (P<0.05), concentration of bile was significantly increased (P<0.05), and contents of bile acid, cholesterol, bilirubin, phospholipid in liver and gallbladder bile were significantly increased in Cldn-2-/- mice (P<0.05). Conclusions: Claudin-2 regulates paracellular water flow which is required for proper regulation of bile composition and flow. Dysregulation of this process increases the susceptibility to cholesterol gallstone related diseases in mice.

Effect of Hic-5 gene knockout on NF-κB/p65 expression and CCl4-induced liver fibrosis degree in mice / 临床肝胆病杂志

@#ObjectiveTo investigate the effect of Hic-5 gene knockout on NF-κB/p65 expression and liver fibrosis. MethodsTen wild-type male C57BL/6 mice were randomly divided into wild-type control group (WT-Control group with 5 mice) and wild-type experimental group (WT-CCl4 group with 5 mice), and ten male C57BL/6 mice with Hic-5 gene knockout were randomly divided into Hic-5 knockout control group (Hic-5 KO-Control group with 5 mice) and Hic-5 knockout experimental group (Hic-5 KO-CCl4 group with 5 mice). The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. Picrosirius red staining was used to observe collagen deposition in liver tissue. Immunohistochemistry was used to measure the expression of alpha-smooth muscle actin (α-SMA) and p65 protein, and real-time quantitative PCR was used to measure the mRNA expression of α-SMA, collagen 1, and p65 in liver tissue. The primary hepatic stellate cells of mice were isolated and stimulated with different concentrations of TGF-β1, and then real-time quantitative PCR was used to measure the mRNA expression of α-SMA, collagen 1, and p65 in primary hepatic stellate cells. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsPicrosirius red staining showed that compared with the WT-CCl4 group, the Hic-5 KO-CCl4 group had a significant reduction in collagen fibers in liver tissue (P＜0.001). Measurement of serum ALT and AST showed that there were significant differences in ALT and AST between the WT-Control group, the WT-CCl4 group, the Hic-5 KO-Control group, and the Hic-5 KO-CCl4 group (F=22.85 and 25.15, both P＜0.001), and the Hic-5 KO-CCl4 group had significantly lower serum levels of ALT and AST than the WT-CCl4 group (both P＜0.05). Immunohistochemistry showed that there were significant differences in the expression levels of α-SMA and p65 protein in liver tissue between the WT-Control group, the WT-CCl4 group, the Hic-5 KO-Control group, and the Hic-5 KO-CCl4 group (F=207.10 and 98.16, both P＜0.001), and the Hic-5 KO-CCl4 group had significantly lower expression of α-SMA and p65 protein in liver tissue than the WT-CCl4 group (both P＜0.01). The results of real-time quantitative PCR showed that there were significant differences in the relative mRNA expression of α-SMA, collagen 1, and p65 in liver tissue between the WT-Control group, the WT-CCl4 group, the Hic-5 KO-Control group, and the Hic-5 KO-CCl4 group (F=41.62, 13.93, and 98.16, all P＜0.001), and the Hic-5 KO-CCl4 group had significantly lower relative mRNA expression of α-SMA, collagen 1, and p65 in liver tissue than the WT-CCl4 group (all P＜0.05). After the primary hepatic stellate cells were stimulated by TGF-β1 at concentrations of 0, 5, and 10 ng/ml, there were significant differences in the relative mRNA expression of α-SMA, collagen 1, and p65 between the WT 0 ng/ml group, the WT 5 ng/ml group, the WT 10 ng/ml group, the KO 0 ng/ml group, the KO 5 ng/ml group, and the KO 10 ng/ml group (F=53.9, 75.82, and 52.41, all P＜0.001), and the Hic-5 KO group had significantly lower relative mRNA expression of α-SMA, collagen 1, and p65 than the WT group (all P＜0.01). ConclusionHic-5 knockout inhibits NF-κB/p65 expression and hepatic stellate cell activation and alleviates CCl4-induced liver fibrosis.

Programmed Cell Death 5 Provides Negative Feedback on Cardiac Hypertrophy Through the Stabilization of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a Protein.

PDCD5 (programmed cell death 5) is ubiquitously expressed in tissues, including the heart; however, the mechanism underlying the cardiac function of PDCD5 has not been understood. We investigated the mechanisms of PDCD5 in the pathogenesis of cardiac hypertrophy. Cardiac-specific PDCD5 knockout mice developed severe cardiac hypertrophy and impaired cardiac function, whereas PDCD5 protein was significantly increased in transverse aortic constriction mouse hearts and phenylephrine-stimulated cardiomyocytes. Overexpression of PDCD5 inhibited phenylephrine-induced cardiomyocyte hypertrophy, and knockdown of PDCD5 induced cardiomyocyte hypertrophy and aggravated phenylephrine-induced hypertrophy. The expression of PDCD5 protein was regulated by NFATc2 (nuclear factor of activated T cells c2) during hypertrophy. SERCA2a (sarco/endoplasmic reticulum Ca2+-ATPase 2a) expression was decreased in PDCD5-deficient mouse hearts because of increased ubiquitination. PDCD5-deficient cardiomyocytes displayed decreased calcium uptake rate, slowed decay of Ca2+ transients, decreased calcium stores, and diastolic dysfunction. Moreover, reintroduction of PDCD5 in PDCD5-deficient mouse hearts reserved SERCA2a protein, suppressed NFATc2 protein, and rescued the hypertrophy and cardiac dysfunction. Our results revealed that PDCD5 is a novel target of NFATc2 in the hypertrophic heart and provides negative feedback to protect the heart against excessive hypertrophy via the stabilization of SERCA2a protein.

Expermental study on hepatic ischemia and reperfusion injury in Rag1 and Rag2/IL2rγ knockout mice / 国际外科学杂志

Objective To explore the effect of lymphocytes on the innate immune cells in Rag1 and Rag2/IL2rγ gene knockout mice after hepatic ischemia and reperfusion injury (HIRI).Methods C57BL/6 mice (n =10),Rag1 knockout mice (n =10) and Rag2/IL2rγ knockout mice (n =10) were respectively divided into sham group and hepatic ischemia-reperfusion injury group by simple randomization,5 mice in each group.By using the model of hepatic ischemia-reperfusion injury in mice,changes of intrahepatic immune cells were detected by flow cytometry.Hepatic ischemia and reperfusion injury and changes of intrahepatic cell subsets were observed in immune system-deficient mice,both Rag1 and Rag2/IL2rγ knockout.Measurement data were expressed as ((x) ±s),and analyzed using independent samples t test.Results Flow cytometry results showed that immune cells,including NK cells,NKT cells,CD4+T cells,DNT cells,Kupffer cells,BMMs and neutrophils were increased after HIRI.Compared with sham group,Rag1 knockout mice displayed markedly increased proportion of Kupffer cells,BMMs and neutrophils after HIRI.And decreased serum ALT levels [from (1 776.25 ± 219.37) U/L to (932.33 ±58.77) U/L,P=0.003,t =7.350] and less hepatocellular necrosis were exhibited in Rag1 knockout mice after HIRI,comparing to C57BL/6 HIRI group.In addition,increased neutrophils were still observed in Rag2/IL2rγ knockout mice after HIRI,without increased proportion of Kupffer cells and BMMs.Compared with Rag1 knockout mice,ALT levels were further decreased from (932.33 ± 58.77) U/L to (309.00 ± 163.53) U/L (P=0.002,t =6.182) in Rag2/IL2rγ knockout mice.Conclusion Both Rag1 and Rag2/IL2rγ knockout mice exhibite less liver injury after HIRI comparing with C57BL/6 mice,indicating that T cells and NK cells aggravate the liver injury.Moreover,T cells do not affect the recruitment of Kupffer cells,BMMs and neutrophils,but regulate the recruitment of NK cells,while NK cells contribute to the activation of Kupffer cells and BMMs,but not neutrophil influx.