Role of umbilical cord mesenchymal stem cell-derived exosomes in acute skin wound healing / 中华皮肤科杂志

Objective:To investigate the role of umbilical cord mesenchymal stem cell-derived exosomes (ucMSC-exos) in acute skin wound healing in mice.Methods:ucMSC-exos were extracted by ultracentrifugation, and identified by transmission electron microscopy, Western blot analysis of exosome surface markers CD63 and TSG101, and particle size analysis. Firstly, in vitro cultured third- to fifth-passage human skin fibroblasts (HSF) were incubated with high-glucose Dulbecco′s modified Eagle′s medium (DMEM) containing 0, 1 and 2 μg/ml exosome suspension for 24 hours (negative control group, 1- and 2-μg/ml groups, respectively) , and cell counting kit-8 (CCK8) assay was performed to evaluate the effect of ucMSC-exos on the proliferative activity of HSF. Secondly, 24 male BALB/c mice aged 8 weeks were selected to construct a mouse model of full-thickness skin wound, and then divided into ucMSC-exos group and phosphate-buffered saline (PBS) group by using a random number table to be subcutaneously injected with exosome suspension and PBS respectively at multiple equidistant sites located about 1 mm apart from the wound edge. On days 0, 4, 7, 10 and 14 after operation, the wounds in mice were observed, and the percentage of residual wound area was calculated in the above two groups. On days 7 and 14 after operation, wound tissues were resected and subjected to hematoxylin and eosin (HE) and Masson staining to observe structural changes of skin tissues. On day 14 after operation, wound tissues were collected in the two groups, and real-time quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of type Ⅰ collagen, fibronectin and vascular endothelial growth factor, respectively. Statistical analysis was carried out by using one-way analysis of variance, least significant difference- t test, two-way repeated measures analysis of variance and unpaired t-test. Results:Under the transmission electron microscope, the ucMSC-exos were oval in shape with a diameter of about 100 nm; Western blot analysis showed positive expression of ucMSC-exos surface proteins CD63 and TSG101; particle size analysis showed that 96.2 % of the ucMSC-exos had diameters of 30 - 150 nm. CCK8 assay showed that the relative proliferative activity of HSF was significantly higher in the 1- and 2-μg/ml groups (0.97 ± 0.05, 1.08 ± 0.07, respectively) than in the negative control group (0.71 ± 0.04; t = 2.00, 7.05, respectively, both P < 0.05) , and significantly higher in the 2-μg/ml group than in the 1-μg/ml group ( t = 5.09, P < 0.05) . On days 4, 7, 10 and 14 after operation, the percentage of residual wound area was significantly lower in the ucMSC-exos group than in the PBS group (all P < 0.05) . HE and Masson staining showed increased numbers of hair follicles, glands and granulation tissues, more neovascularization, and neater arrangement of collagens in neonatal skin tissues of the mice in the ucMSC-exos group compared with the PBS group. qRT-PCR and Western blot analysis showed significantly increased mRNA and protein expression of type Ⅰ collagen, fibronectin and vascular endothelial growth factor in the ucMSC-exos group compared with the PBS group (all P < 0.01) . Conclusion:Subcutaneous injections of ucMSC-exos can promote acute skin wound healing in mice, likely by promoting the synthesis of extracellular matrix and vascular endothelial growth factor in wound tissues of mice and proliferation of HSF.

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Genetically encoded Ca(2+) indicators (GECI) are important for the measurement of Ca(2+) in vivo. GCaMP2, a widely-used GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5-fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca(2+) binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca(2+) signals in intact organs to facilitate the further improvement of GCaMP sensors.

Effect of trimebutine maleate on ion channels in colon smooth muscle cell of cold restraint stress induced rats / 中华消化杂志

Objective To investigate the effects of trimebutine maleate (TM) on the expression of large conductance calcium-activated potassium channel (BKCa) and ryanodine receptors (RyR)channels at mRNA and protein level in colonic smooth muscle cell of cold restraint stress(CRS)induced rats.Methods A total of 24 Wistar rats were divided into CRS group,CRS with TM group and control group equally.The rats of CRS group were gavaged with 0.9％NaCl (6 ml/kg) daily; the rats of CRS with TM group were gavaged with 15 g/L TM (6 ml/kg) daily and activity was restricted in wire cage at 4 ℃ for two hours,continuously for five days.The rats of control group were gavaged with 0.9 ％ NaCl (6 ml/kg) once without CRS.The amount and characteristics of stool of rats in each group were observed.The colonic smooth muscle was isolated to detect the expression of BKCa and RyR at mRNA and protein level by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot.Results The median of rats defecation particles of CRS group was six,control group was one and CRS with TM group was five.Compared with control group,the defecation appearance of CRS group and CRS with TM group was looser and wetter observed by naked eyes.Compared with control group,there was no obvious pathological changes in CRS and CRS with TM group.There was no significant difference in the mRNA expression of BKCa and RyR channels between control group and CRS group.Compared with control group,the BKCa expression at mRNA level of CRS with TM group increased 1.45 fold.Compared with control group,the RyR2 expression at mRNA level of CRS with TM group increased 1.32 fold.Compared with control group,the BKCa expression at protein level of CRS with TM group increased 1.39 fold,and there was no RyR2 expression band at protein level.Conclusion TM might affect colonic smooth muscle contraction through the upregulation of BKCa expression at mRNA and protein level and RyR expression at mRNA level.

Generation of Calhm1 knockout mouse and characterization of calhm1 gene expression

Alzheimer's disease (AD) is the most common neurodegenerative disease among elderly people worldwide. Several genes have been validated to be associated with AD, and calcium homeostasis modulator 1 (Calhm1) is the latest suspected one. To investigate the biological and pathological function of Calhm1 systematically, we generated a Calhm1 conventional knockout mouse. However, both the male and female of elderly Calhm1 knockout (KO) mice showed similar ability to their wild type littermates in spatial learning and memory retrieving. Surprisingly, we found that Calhm1 mRNA could not be detected in mouse brains at different ages, although it is expressed in the human brain tissues. We further found that CpG islands (CGIs) of both mouse and human Calhm1 were hypermethylated, whereas CGI of mouse Calhm2 was hypomethylated. In addition, transcriptional active marker H3K4Di occupied on promoters of human Calhm1 and mouse Calhm2 at a considerable level in brain tissues, while the occupancy of H3K4Di on promoter of mouse Calhm1 was rare. In sum, we found that mouse Calhm1 was of rare abundance in brain tissues. So it might not be suitable to utilize the knockout murine model to explore biological function of Calhm1 in the pathogenesis of AD.

ERp44 C160S/C212S mutants regulate IP3R1 channel activity

Previous studies have indicated that ERp44 inhibits inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release (IICR) via IP(3)R(1), but the mechanism remains largely unexplored. Using extracellular ATP to induce intracellular calcium transient as an IICR model, Ca(2+) image, pull down assay, and Western blotting experiments were carried out in the present study. We found that extracellular ATP induced calcium transient via IP(3)Rs (IICR) and the IICR were markedly decreased in ERp44 overexpressed Hela cells. The inhibitory effect of C160S/C212S but not C29S/T396A/ΔT(331-377) mutants of ERp44 on IICR were significantly decreased compared with ERp44. However, the binding capacity of ERp44 to L3V domain of IP(3)R(1) (1L3V) was enhanced by ERp44 C160S/C212S mutation. Taken together, these results suggest that the mutants of ERp44, C160/C212, can more tightly bind to IP(3)R(1) but exhibit a weak inhibition of IP(3)R(1) channel activity in Hela cells.

The effects of IHC-72 on arrhythmia and lipid peroxides of ischemia and reperfusion rat hearts in vivo / 中国药理学通报

Acute myocardial ischemia and reperfusion led to arrhythmias and increase of glutamic oxalacetic transaminase (GOT),lactate dehydrogenase (LDH),non-csterified fatty acid (FFA) and malondialdehyde (MDA) content, and decrease of superoxide dismutase (SOD) activity in rats. 3, 6-di-methylamino-dibenzopyri-odonium edetate (IHC-72) 5 mg?kg-1 iv significantly reduced the incidence of ventricular tachy-cardia (VT) and ventricular fibrillation(VF) ,shortened the duration of arrhythmias induced by reperfusion, decreased the release of GOT, LDH and FFA, obviously reduced the MDA content,and effectively protected SOD activity in ischemia-reperfused rat hearts. Our experimental results suggested that IHC-72 had protective effects on ischemia-reperfusion injury rat hearts in vivo. The mechanism of the protection might be associated with the inhibition of cellular lipid peroxidation.