Transplantation of bone marrow mesenchymal stem cells improves motor function in a DMD mouse model

AIM: To observe amelioration of motor function in a Duchenne muscular dystrophy (DMD) mouse model (dko mice) after transplantation of bone marrow mesenchymal stem cells (MSCs). METHODS: Passage fifth MSCs cultured in vitro were transplanted into dko mice by tail vein, motor functions of experimental mice and matched control mice, including traction, rotating rods, rotated wheel, upside down, turning over and walking (all were recorded by Sony digital camera) were tested 15 weeks after transplantation. The fluorescent expression of dystrophin and utrophin in gastrocnemius muscle tissue of dko mice was detected by SABC-Cy3, and average optical density of positive fibers was calculated. RESULTS: MSCs grew in colony over passage third, and there was low immunologic reaction by vein transplantation. There was dystrophin and utrophin fluorescent expression in sarcolemma of dko mice 15 weeks after transplantation, but no any fluorescent expression in controls. There was significant difference in fluorescent average optical density of positive fibers between two groups (P＜0.05). Amelioration of motor functions in dko mice was found 15 weeks after MSCs transplantation compared with the control mice (P＜0.05). CONCLUSION: Transplantation of MSCs ameliorates the positive and passive motor functions of dko mice.     

Effects of PFOA exposure on inflammatory mediators IL-4, IFN-γ and glucocorticoid receptor in asthmatic mice

AIM: To investigate the effects of perfluorooctanoic acid (PFOA) exposure on the changes of asthmatic mouse airway inflammation, inflammatory mediators interleukin-4 (IL-4) and interferon-γ (IFN-γ) in serum, and glucocorticoid receptor (GR) expression in the lung tissue.METHODS: BALB/c mice (n=30) were randomly divided into 5 groups:normal control (C) group, asthma (A) group, asthma+low-dose PFOA (AP10) group, asthma+ mode-rate-dose PFOA (AP50) group and asthma+high-dose PFOA (AP100) group. Asthma model and PFOA exposure model of mice were established according to the grouping. The animals were sacrificed and their lungs were collected for HE staining, transmission electron microscopy, Western blot and immunohistochemical staining. ELISA was applied to detect the levels of IL-4 and IFN-γ in the serum.RESULTS: HE staining of the lungs showed that the asthmatic mice, compared with the normal control mice, had obvious mucus secretion around the airways and infiltration of inflammatory cells around airways and blood vessels, and the effects were much more marked in AP groups. Ultrastructural alteration of the lung tissues in the asthmatic mice were indicated by transmission electron microscopy. Compared with C group, the results of ELISA in A group and AP groups proved that IL-4 in the serum was increased and IFN-γ was decreased significantly (P<0.05). Compare with A group, IL-4 was significantly increased and IFN-γ was decreased in AP100 group (P<0.05), and no difference of those between AP10 group and AP50 group was found. The results of Western blot indicated that GR protein expression in the asthmatic mice were decreased compare with the normal mice (P<0.05), and no difference of that among A group and AP groups was observed. Immunohistochemical staining manifested that GR protein was mainly located in the cytoplasm of bronchial columnar epithelial cells, airway smooth muscle cells and vascular smooth muscle cells.CONCLUSION: Acute airway PFOA exposure in asthmatic mice dose-dependently exacebates lung inflammation by inducing Th2 type immune responses, promotes infiltration of inflammatory cells and mucus secretion around the airways and blood vessels, and destroys the ultrastructure of the lung tissues.     

Breeding of APPSWE transgenic mice and gene identification of filial generation

AIM: To explore the ideal breeding way of APPSWE transgenic mouse (Tg2576) and identify filial generation mice with APPSWE gene for laying a foundation of AD research. METHODS: (1) Three different copulation ways were adopted to observe the survival rate and positive rate with APPSWE gene of filial generation mice. (2) The genome DNA was extracted from the tails of filial generation mice and PCR method was employed to amplify the APPSWE gene fragment. The gene type was observed by electrophoresis.(3) PCR product was inserted in pGEM-T vector to detect its gene sequence. RESULTS: The male Tg2576 mice mated with female Tg2576, and four litters were reproduced, all of them died during two days. The male Tg2576 mice mated with female C57BL, and the survival rate of their filial generation was 81% and positive rate with APPSWE gene was 43.3%. The male C57BL mice mated with the female Tg2576,and the survival rate and positive rate with APPSWE gene of filial generation mice were 82.4% and 21.9%, respectively. By Wilcoxon's Rank Sum Test, there were no significant different between the survival rates of filial generation mice by two breeding way (P＞0.05), but there were significant different between the positive rates with APPSWE gene (P＜0.05). Electrophoresis result showed the molecular weight of PCR product was 428 bp and was in accordance with that of APPSWE gene fragment. By gene sequencing, gene sequence of PCR product was verified as same as APPSWE gene double mutant. CONCLUSION: It is ideal way of breeding filial generation mice with APPSWE gene that male Tg2576 mouse mate with female C57BL. PCR technique can identify the filial generation with APPSWE gene precisely, and offer an ideal animal model for further research on AD.     

Therapeutic effect of emodin on constipation induced by loperamide in mice

AIM: To study the therapeutic effect of emodin on loperamide-induced constipation in mice. METHODS: The constipation model of mice was established by lopebutamine treatment, and the effects of emodin on defecation frequency, fecal water content and intestinal transit time of the mice during the observation period were detected. Inflammatory infiltration in colon tissue of the mice was observed by HE staining. Serum nitric oxide (NO) content was detected by commercially available kit. The expression of vasoactive intestinal peptide receptor 1 (VIPR1) and 5-hydroxytryptamine type 4 receptor (5-HT₄ receptor) in mouse colon was determined by immunohistochemistry. The effects of emodin on the expression of transient receptor potential cation channel subfamily V member 1 (TRPV1), glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), nitric oxide synthase (NOS), c-Kit and their ligand stem cell factor (SCF) were detected by Western blot. RESULTS: Emodin significantly increased the number of defecation and fecal water content in the mice during the observation period, and significantly reduced the intestinal transit time and serum NO level in the mice (P<0.05). The results of HE staining showed that emodin significantly reduced the infiltration of colonic inflammation induced by loperamide. Emodin can significantly reduce the increase in VIPR1 induced by loperamine and increased the expression of 5-HT₄ receptor (P<0.01). Emodin increased the expression levels of GDNF and BDNF, reduced the expression levels of TRPV1 and NOS in the colon tissues of loperamine-induced constipation in mice, and significantly increased the expression of c-Kit and SCF (P<0.05 or P<0.01). CONCLUSION: Emodin promotes the defecation behavior of mice with loperamine-induced constipation by increasing intestinal peristalsis and activating a series of smooth muscle contraction-related factors.     

Effect of garlicin on viral myocarditis in mice

AIM: To investigate the effect of garlicin on mouse viral myocarditis and to explore the possible mechanisms. METHODS: Forty BALB/c mice were randomly divided into 4 groups: control group, viral myocarditis group, low-dose garlicin group and high-dose garlicin group. The morphological changes of the myocardium were observed with HE staining. The levels of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and creatine kinase (CK) were measured by colorimetric method. The protein levels of Bcl-2, Bax, cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP and cleaved-caspase-8 in the myocardial tissues were determined by Western blot. RESULTS: The morphological observation with HE staining showed that garlicin inhibited the necrosis of the myocardium and infiltration of inflammatory cells. Compared with viral myocarditis group, garlicin dose-dependently decreased the levels of LDL, AST and CK. Moreover, garlicin treatment decreased the protein levels of Bax, cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP, accompanied with an increase in Bcl-2 expression. However, garlicin had no effect on the protein level of cleaved-caspase-8. CONCLUSION: Garlicin effectively attenuates the myocardial damage in mice with viral myocarditis through inhibition of intrinsic apoptosis pathway.     

Skeletal muscles of mdx mice were damaged after overload exercise

AIM:To observe the effects of overload exercise on skeletal muscles in X-linked muscular dystrophy(mdx) mice.METHODS:Mdx mice and C57 mice were carried out swimming and hanging tail movement tests (mdx mice as control did not exercise). It lasted for 13 minutes each time per day, and lasted 3 days. Evans blue was injected into tail vain. The mice were killed the next day, and the hind limbs were taken photographs after skins were flayed. The gastrocnemius muscles and diaphragms cryostat sections were made. Under a fluorescence microscope, Evans blue staining was seen. Then the sections were tested by routine HE staining, the histological change of muscles was analyzed under a light microscope.RESULTS:Many blue colored longitudinal lines were observed in skeletal muscles of mdx mice, whereas they were hardly seen in control mdx and C57 mice. Under a fluorescence microscope, some muscle fibers of mdx mice were stained with Evans blue, few muscle fibers of control mdx mice were stained, and C57 mice were not. Under a light microscope, HE staining of muscles showed some degenerated muscle fibers became round in shape and the myonuclei became condensed, or necrotic fibers had amorphous structures, most of them in the degenerated and necrotic fibers of diaphragms C57 mice did not have these changes.CONCLUSION:Overload exercise did harm to skeletal muscles of mdx mice; Vital staining with Evans blue is useful not only for distinguishing degenerating muscle fibers, but also for studying the degeneration process in dystrophin-deficient muscle.     

朱顶红无菌苗叶片高效再生体系

以朱顶红（Hippeastrum vittatum）叶片为外植体进行离体培养，具有取材方便、试材充足、成本低等优势，但叶片诱导再生率极低，是朱顶红离体培养的一大难题。本试验中分别以‘花孔雀’和‘黑天鹅’朱顶红无菌苗叶片为外植体，探究了不同植物生长调节剂和不同取材部位对不定芽诱导和继代增殖的影响。结果表明：最佳外植体为 MS 培养基中培养 10 d 形成的幼嫩叶片基部（0.5 cm），在光照 16 h · d-1（光照强度 36 μmol · m-2 · s-1）下，不定芽诱导的最适培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 2 mg · L-1 TDZ，两个品种的不定芽均以间接途径发生，其中‘花孔雀’在培养 40 d 后形成愈伤组织，55 d形成不定芽，诱导率可达 69.44%；‘黑天鹅’在培养 45 d 后形成愈伤组织，65 d 形成不定芽，诱导率达到 66.67%；最适体细胞胚诱导培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 PIC，‘花孔雀’和‘黑天鹅’的诱导率分别达到 66.67%和 63.89%；最佳不定芽增殖培养基为 MS + 2 mg · L-1 6-BA + 1 mg · L-1 NAA + 1 mg · L-1 TDZ，‘花孔雀’和‘黑天鹅’的增殖系数分别达到 4.67 和 3.46；在不添加植物生长调节剂的 MS培养基中进行生根培养，30 d 后两个品种的生根率均达到 100%；将生根培养 30 d 的小植株转移至室温条件下放置 3 d，摘去封口膜再驯化 3 d 后，移栽至经高温消毒的草炭︰蛭石（体积比）为 1︰1 的基质中，成活率达到 100%     

Experimental treatment of the model mice of Duchenne muscular dystrophy by bone marrow transplantation

AIM: To detect dystrophin expression in skeletal muscles of mdx mice after bone marrow transplantation (BMT), and to evaluate the effect of BMT on Duchenne muscular dystrophy (DMD). METHODS: Bone marrow cells were cultured for three days, and then transplanted into mdx mice irradiated lethally through tail veins. After 4 and 6 months, dystrophin expression on myocytes membranes in mdx mice was detected by fluorescent immunohistochemical staining. The centrally nucleated fibers (CNF) were calculated by HE staining, and the physiologic parameters measured and the motor function detected by traction test, rotating rods test and rotating wheels test were also observed. RESULTS: Until 4 and 6 months after BMT, dystrophin was expressed partly on myocytes membranes in mdx mice, and the ratio of CNF decreased, physiologic functions improved, the motor ability reinforced in treated group. CONCLUSION: After BMT, marrow stem cells settled in injured skeletal muscles and bone marrow, then differentiated into myocytes with dystrophin expression and caused the improvement of pathology, physiology and motor function in treated group finally. These results give a powerful proof for the treatment of DMD with BMT.     

Establishment of a transgenic heterozygous mouse model of ApcMin/+ precancerosis of colorectal cancer with p110δ mutation

AIM：To establish a transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation in the C57BL/6J background for serving the studies on colorectal cancer research mediated by p110δ. METHODS：The transgenic heterozygous mice were generated by crossing in p110δD910A/D910A mouse and ApcMin/+ mouse, and the genotype was detected by PCR. Compared with ApcMin/+ mice, transgenic heterozygous mice (ApcMin/+; p110δD910A/D910A)were counted, and the number and size of intestine polyps were analyzed after methylene blue staining. The intestinal tissue structure was assessed by HE staining. RESULTS：The transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation was established. The number and size of polyps in the transgenic heterozygous mice were declined. CONCLUSION：A transgenic heterozygous mouse model of precancerous lesions of colorectal cancer with p110δ mutation was successfully established. The initial phenotype of intestinal tumors in transgenic mice was observed. This model will greatly contribute to the related research of colorectal cancer in mice.     

Effect of wogonoside on inflammatory response in mice with viral myocarditis induced by Coxsackie virus B3

AIM: To investigate the effect of wogonoside on the inflammatory response of mice with Coxsackie virus B3 (CVB3)-induced myocarditis and its possible regulatory mechanism. METHODS: A mouse model of viral myocarditis was constructed by infecting BALB/c mice with CVB3. BALB/c mice (n=40) were randomized into 4 groups: normal group, CVB3-induced viral myocarditis group, CVB3-induced viral myocarditis combined with wogonoside treatment group and CVB3-induced viral myocarditis combined with wogonoside plus AKT agonist treatment group. All the mice were sacrificed 7 days after treatment. In the first 3 groups, HE staining was applied to detect the infiltration of inflammatory cells in the myocardium, ELISA was applied to detect the serum levels of interleukin-1β (IL-1β) and IL-6, while Western blot was applied to detect the protein expression of inflammatory factors and the activation of AKT/NF-κB pathway. Inaddition, the activation of AKT/NF-κB pathway in the 4 groups was detected by Western blot analysis. RESULTS: HE staining showed that there was a large amount of inflammatory cell infiltration in the myocardium of CVB3-induced viral myocarditis mice, as compared with the normal group, which was significantly reduced by wogonoside treatment (P<0.05). The serum levels of IL-1β and IL-6 in the mice after CVB3 infection were significantly higher than those in normal group (P<0.05), which was also significantly reduced by wogonoside treatment (P<0.05). Western blot analysis indicated that wogonoside treatment significantly reduced the expression of inflammatory factors IL-1β and IL-6, and the phosphorylation of AKT/NF-κB pathway-related proteins in the myocardial tissue (P<0.05). After administration of AKT agonist, the inhibitory effect of wogonoside on NF-κB phosphorylation and inflammatory factors expression was significantly eliminated (P<0.05). CONCLUSION: Wogonoside attenuates the inflammatory response of mice with viral myocarditis by inhibiting the AKT/NF-κB pathway.     

Influence of Jiaomu oil A2 on eosinophil manifold and CD34+ with marrow granule system mobilization in bronchia asthma mice

AIM: To observe the influence of Jiaomu oil A2 on eosinophil manifold and CD34+ with marrow granule system mobilization in bronchial asthma mice. METHODS: The asthmatic mouse model was established by sensitization and challenge of the animals with 20% Al(OH)3+10% ovalbumen (OVA). After the mice were excitated for 10 d and giving medical therapy at the same time, the mice were executed, the bronchial-alveolar lavage inflammatory cells and the hemocytopoiesis cells were examined using Wrish-Giemsa staining. The expressions of interleukin-5 (IL-5) and eotaxin (EON) in lung tissue and marrow were examined by in situ hybridization. The expression of IL-5 and EON albumen in lung tissue and marrow were detected by immunohistochemistry. The inflammatory cell infiltration and CD34+ cells in lung tissue were also observed by HE and immunofluorescence staining. RESULTS: Both Jiaomu oil A2 and prednisone significantly attenuated the pathological process degree of bronchial inflammatory reaction such as tissue swelling, reconstruction, hyperplasia, and epithelial cell shedding, and inhibited eosinophil count and infiltration caused by stimulation of allergic effect. Moreover, the two drugs markedly lessen the eosinophil density in tracheal surrounding tissue and marrow in asthma mice and depressed the differentiation of marrow myelocyte to eosinophil. Finally, the apparent decrement of CD34+IL-5, CD34+IL-5R, CD34+CCR-3 cells in bronchial tissue and marrow showed some relationship with the downregulation of IL-5, IL-4, GM-CSF in lung tissue. CONCLUSION: The effect of Jiaomu oil on airway inflammation in asthma mice is associated with inhibiting the mobilization of eosinophil and marrow granule system.     

Suramin alleviates hypertrophic scar in mice by inhibiting fibroplasia and inflammatory response

AIM: To investigate the therapeutical effect of suramin on hypertrophic scar (HPS) and its mechanism. METHODS: After the mouse model of HPS was established by mechanical stretching, the suramin solution at low dose (5 mg/kg) and high dose (10 mg/kg) was applied onto the scar site caused by mechanical load in mice by transdermal administration once a day for 10 d. The degree of scar hyperplasia was observed by macroscopy. The scar cross-sectional area and scar elevation index in the HPS tissues were evaluated by hematoxylin-eosin (HE) staining. The expression levels of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) in HPS tissues were detected by immumohistochemical staining. The expression level of α-smooth muscle actin (α-SMA) in HPS tissues was detected by immunofluorescence staining, RT-qPCR and Western blot. The levels of tumor necrosis factor-α (TNF-α), IL-6, IL-10 and TGF-β1 in the HPS tissues were measured by ELISA. RESULTS: Macroscopic observation showed that the surface areas of scar in the HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). HE staining results showed that the scar cross-sectional area and the scar elevation index of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.05 or P<0.01). The results of immunofluorescence staining, RT-qPCR and Western blot showed that the number of α-SMA positive cells and the mRNA and protein expression of α-SMA in scar tissues of HPS mice after treatment with suramin at low and high doses were significantly decreased (P<0.05 or P<0.01). The results of immunohistochemical staining showed that the expression levels of TGF-β1 and IL-6 in scar tissues of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). The results of ELISA showed that the levels of TNF-α, IL-6, IL-10 and TGF-β1 in the scar tissues of HPS mice after treatment with suramin at low and high doses were significantly reduced (P<0.01). CONCLUSION: Suramin inhibits the formation of HPS, which may be related to the inhibition of fibroplasia and reduction of local inflammatory response.     

Role of microRNA-126-5p in myocardial injury induced by doxorubicin

AIM: To observe the expression of microRNA-126-5p during myocardial injury and its role in myocardial cell injury induced by adriamycin (also called doxorubicin, DOX). METHODS: The BALB/c mouse model of DOX-induced acute and chronic myocardial injury was established via intraperitoneal injection of DOX. HE staining was applied to observe the morphological changes of myocardial tissues. Lactate dehydrogenase (LDH) in serum was detected and PowerLab system was used to detect the influence of DOX on the changes of ±dp/dt_max. The expression of microRNA-126-5p in injured myocardial tissues and the H9c2 cells exposed to DOX was detected by real-time PCR. Gain-and loss-of-function experiments were conducted to detect the role of microRNA-126-5p in H9c2 cells treated with DOX on LDH release and caspase-3 activation. RESULTS: In acute and chronic DOX myocardial damage models in mice, HE staining showed disarranged myocardial fibers, dissolved myofibril and inflammatory cell infiltration. Higher serum LDH level and lower ±dp/dt_max in DOX-treated mice than those in normal mice were found. Compared with the normal mice, the expression level of microRNA-126-5p was significant increased in the myocardium with DOX-induced injury. Similarly, the expression level of microRNA-126-5p was significant increased in the H9c2 cells treated with DOX. In addition, over-expression of microRNA-126-5p decreased cell viability and promoted apoptosis, while microRNA-126-5p ablation promoted the viability and inhibited the apoptosis of H9c2 cells. CONCLUSION: The microRNA-126-5p expression is up-regulated in myocardial injury induced by DOX, and microRNA-126-5p inhibits cell viability and promotes apoptosis induced by DOX.     

Impaired function of pancreatic β cells in the R6/2 transgenic mouse model of Huntington's disease

AIM: To study the function of pancreatic β cells in the R6/2 transgenic mouse of Huntingtons disease(HD), and to elucidate the pathogenetic mechanisms underlying diabetes mellitus in transgenic mice of HD. METHODS: By using the R6/2 transgenic mouse model of HD, fasting blood glucose and fasting insulin concentration in plasma of normal and HD mice were detected. Further, HE staining and immunofluorescence technique were used for morphometric analysis of islets in normal and HD mice. RESULTS: In contrast to normal mouse, R6/2 HD mouse showed hyperglycemia and hypoinsulinemia in fasting state. Pancreatic islets morphology showed that islets atrophied and cell number decreased in HD mouse. Poor functional index was observed in these mice, but insulin resistance index was normal. CONCLUSION: Impaired function of pancreatic cells may be the key factor contributing to the pathogenesis of diabetes in the R6/2 transgenic mouse model of HD.     

Anthocyanins attenuates hepatic fibrosis and inflammation in non-alcoho-lic fatty liver disease mice

AIM:To explore the therapeutic effect of anthocyanins from Fructus Acanthophorae on high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) in mice and the potential mechanism. METHODS:NAFLD mouse model was established by high-fat diet, and interferred with anthocyanins. The liver weight, and serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC) and low-density li-poprotein cholesterol (LDL-C) were measured. The liver tissues were staining with HE, Oil Red O and Masson's trichrome. The protein levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and IL-10 in the liver tissues were determined by Western blot. The liver macrophage, white blood cell and mononuclear cell infiltration was detected by immunohistochemical method. The chemokines CCL7 and MCP-1 were also measured by immunohistochemical method. RESULTS:Anthocyanins significantly inhibited the increases in the liver weight, ALT, AST, TG, TC and LDL-C induced by high-fat diet. Anthocyanins attenuated the liver fibrosis and inflammatory cell infiltration caused by high-fat diet, and reduced the levels of inflammatory factors TNF-α, IL-1β, IL-6, IL-10 and inflammatory chemokines CCL7 and MCP-1 in the liver tissues. CONCLUSION:Anthocyanins significantly alleviate non-alcoholic fatty liver disease caused by high-fat diet though reducing inflammatory factors, inflammatory cell infiltration and inflammatory chemokines.     

Role of miR-433 in fibrosis of NIH-3T3 cells and silica-induced pulmonary fibrosis in mice

AIM To study the role and regulatory mechanism of microRNA-433 (miR-433) in fibrosis. METHODS TargetScan was used to predict the potential target genes of miR-433. The changes of miR-433 expression were detected after transforming growth factor β1 (TGF-β1) treatment of mouse embryonic fibroblasts (NIH-3T3 cells) for 24 h. The effects of miR-433 mimic on the expression of p-SMAD2, fibronectin (FN), α-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF) in TGF-β1 treated cells were examined. The effects of miR-433 mimic transfection on the viability and S-phase fraction of NIH-3T3 cells induced by TGF-β1 were detected by CCK-8 assay and flow cytometry. A model of silica-induced pulmonary fibrosis in mice was established, and agomiR-433 was used for intervention. HE staining and Masson staining were used to observe the effect of miR-433 on pulmonary fibrosis in the mice. The expression of α-SMA in lung tissues was detected by immunohistochemistry. RESULTS miR-433 specifically bound to the 3'-UTR of SMAD2 and inhibited its expression at protein and mRNA levels. TGF-β1 down-regulated the expression of miR-433 in NIH-3T3 cells, up-regulated the protein level of p-SMAD2 and the expression of FN, α-SMA and CTGF at protein and mRNA levels, and increased the viability and the number of S-phase cells. miR-433 mimic reversed the effects of TGF-β1 on NIH-3T3 cell viability and S-phase arrest. In a model of silica-induced pulmonary fibrosis in mice, agomiR-433 inhibited the progress of pulmonary fibrosis and reduced the expression of α-SMA in mouse lung tissues. CONCLUSION miR-433 may interfere with TGF-β1/SMAD2 signaling pathway through targeting SMAD2, thus participating in the regulation of fibrosis process.     

Effect of microRNA-7 knockdown on ConA-induced acute liver injury in mice

AIM: To study the effect of microRNA-7 (miR-7) knockdown (KD) on concanavalin A (ConA)-induced acute liver injury (ALI) in mice.METHODS: Wild type (WT) mice and miR-7KD mice were received ConA (30 mg/kg) to induced acute liver injury model by intraperitoneal injection, and the morphological changes, liver weight and weight index were measured 48 h later. The pathological changes of the liver tissues were observed by HE staining. The levels of serum alanine aminotransferase (ALT), IL-4 and IFN-γ were detected by ELISA. The proportional changes of CD4⁺ T cells and the relative levels of IL-4 and IFN-γ were analyzed by flow cytometry.RESULTS: The color of the liver tissue became lighter, and the weight and weight index were changed significantly in miR-7KD mice compared with control group (P<0.05). HE staining showed that the inflammatory cell infiltration was increased in the liver of miR-7KD mice. Moreover, the level of serum ALT was significantly increased (P<0.05). The serum level of IFN-γ elevated significantly (P<0.01), while the IL-4 levels decreased significantly (P<0.01) in the serum of miR-7KD mice. Furthermore, the proportion of CD4⁺ T cells and relative IFN-γ cells increased obviously (P<0.01).CONCLUSION: miR-7 knockdown promotes the pathogenesis of the ConA-induced acute liver injury in mice.     

Cigarette dust particles induced lung function injury of chronic obstructive pulmonary disease in mice

AIM: To investigate the pathogenesis of chronic obstructive disease (COPD) in mice by using nasal drip of cigarette dust particles (DSP) induced pulmonary function damage model.METHODS: BALB/c mice were randomly divided into control group, LPS 100 mg/L group, DSP 0.75 mL/L group, DSP 1.5 mL/L group and DSP 3 mL/L group for 30 days. The method of nasal drip was used for 30 days to establish the COPD model. Rrs, Ers, Crs, Est, Cst, P-3/8Rn, P-3/8G and P-3/8H were measured for evaluating lung function of the mice in each group by the method of FlexiVent. The effect on the increase of airway resistance induced by methacholine (Mach) was determined using main bronchial rings by Myograph method. The HE, Masson and Resorcinol fuchsin staining of mouse tracheas and lung tissues were conducted. RESULTS: Continuous nasal drip with DSP for 30 days increased Rrs, Ers, Est, P-3/8Rn and decreased Crs, Cst, P-3/8G and P-3/8H in the mice. DSP significantly shifted the dose-effect curve of tracheal contraction induced by Mach to the left, increased the sensitivity of the airway to Mach, and significantly increased the maximal contractile airway effect of Mach. Exposure to DSP caused fibrosis of airway subepithelial, deposition of collagen in the airway basement membrane under the reticular plate, induced reticular plate thickening, pulmonary bronchial lumen serious deformation, and the inflammatory cell infiltration in the lung of mice. Significantly increased alveolar wall muscle fibers and collagen fibers were also observed. CONCLUSION: The lung function and pathomorphological changes of COPD mice induced by 30 days nasal drip of cigarette dust particles were similar to those of human COPD.     

Notch1 promotes renal tubulointerstitial fibrosis in renal tissues of diabetic mice by inhibiting autophagy through Akt/mTOR signaling pathway

AIM: To observe the changes of Notch1 expression and autophagy in the renal tissues of diabetic mice, and to explore the regulatory effect of Notch1 on tubulointerstitial fibrosis by inhibiting autophagy in diabetic nephro-pathy. METHODS: The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice), with 8 rats in each group. After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. The protein expression of Notch1 in the renal tubular epithelial cells was observed by immunohistochemical staining. The protein levels of Notch1, PTEN, p-Akt (Thr308), Akt, p-mTOR (Ser2448), mTOR, LC3, P62, collagen type Ⅰ (Col-Ⅰ) and collagen type Ⅲ (Col-Ⅲ) were determined by Western blot. RESULTS: Compared with the db/m mice, the blood glucose, glycosylated hemoglobin, serum creatinine, triglyceride and total cholesterol were increased in the db/db mice (P<0.01). Renal tubular epithelial cell vacuolar degeneration, renal tubular expansion and interstitial inflammatory cell infiltration in db/db mouse renal tissues with HE staining were observed. The images of Masson staining showed collagenous fiber-like substance deposition in the glomerular capillaries and renal interstitium, and disarrangement of tubular structure in the renal tissues of db/db mice. The protein expression levels of PTEN and LC3-Ⅱ were decreased (P<0.01 or P<0.05), while the protein levels of Notch1, P62, p-mTOR (Ser2448), p-Akt (Thr308), Col-I and Col-III were increased in the db/db mice as compared with the db/m mice (P<0.01). However, no significant change of total mTOR and Akt proteins between the 2 groups was found. CONCLUSION: Notch1 protein expression was increased, PTEN expression was significantly reduced, Akt/mTOR pathway was activated, autophagy was inhibited, and fibrosis was aggravated in the renal tissues of the diabetic mice.     

Human umbilical mesenchymal stem cell-derived exosome attenuates cardiac fibrosis in diabetic mice

AIM: To explore the protective effects of exosome secreted by human umbilical mesenchymal stem cells on cardiac fibrosis in diabetic mouse model. METHODS: Male C57BL/6 mice at 6~8 weeks of age were divided into 3 groups randomly:control group, diabetes mellitus (DM) group and DM+exosome group. To develop mouse DM mo-del, the mice were fed with high-fat diet for 5 weeks, followed by intraperitoneal injection of 45 mg/kg streptozocin once a week for 5 weeks. It was considered as a successful DM model that the blood glucose of the mice was ≥ 16.7 mmol/L. The mice in DM+exosome group were injected with exosome via tail vein. The mice in other 2 groups were injected with saline at the same volume. The heart function was evaluated by color Doppler echocardiography for small animals. The blood samples were collected from abdominal aortas. The blood glucose and non-esterified fatty acids were measured by biochemical colorimetric assay. HE staining was performed to observe the structural changes of myocardial fibers, and Masson staining was used to observe the cardiac fibrosis. RESULTS: The results of echocardiography showed that left ventricular end-diastolic dimension (LVIDd) and left ventricular end-systolic dimension (LVIDs) of diabetic mice were larger than those in control mice (P<0.05 and P<0.01, respectively). The ejection fraction (EF) and fractional shortening (FS) decreased in the diabetic mice (P<0.01). Exosome treatment significant decreased the LVIDs (P<0.01), but increased the EF and FS (P<0.01). The blood glucose and non-esterified fatty acids were significantly increased in the diabetic mice. The injection of the stem cell exosome significantly decreased the blood glucose and non-esterified fatty acids (P<0.01). HE staining observation showed that cardiomyocyte hypertrophy and fragmentation of cardiomyocyte in DM group were more se-rious than those in control group. Masson staining showed that the area of fibrosis in DM group was larger than that in control group (P<0.01), but that in DM+exosome group was reduced (P<0.01). CONCLUSION: Exosome secreted by human umbilical mesenchymal stem cells protects the DM model mice from cardiac fibrosis.