AIMTo investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β<sub>4</sub> (AAV-Tβ<sub>4</sub>) on murine colitis via intracolonic a...AIMTo investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β<sub>4</sub> (AAV-Tβ<sub>4</sub>) on murine colitis via intracolonic administration.METHODSAAV-Tβ<sub>4</sub> was prepared and intracolonically used to mediate the secretory expression of Tβ<sub>4</sub> in mouse colons. Dextran sulfate sodium (DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to establish a mouse colitis model resembling Crohn’s disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tβ<sub>4</sub> on colitis. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis and proliferation were detected by TUNEL assay and immunochemistry, respectively.RESULTSRecombinant AAVs efficiently delivered LacZ and Tβ<sub>4</sub> into the colonic tissues of the mice, and AAV-Tβ<sub>4</sub> led to a strong expression of Tβ<sub>4</sub> in mouse colons. In both the DSS and TNBS colitis models, AAV-Tβ<sub>4</sub>-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tβ<sub>4</sub> significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tβ<sub>4</sub> also modulated colonic TNF-α, IL-1β and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice.CONCLUSIONTβ<sub>4</sub> exerts a protective effect on murine colitis, indicating that AAV-Tβ<sub>4</sub> could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.展开更多
Chemically based rodent models are used to assess the positive effects promoted by foods and gut microbiota on gut health. Lectins with enzymatic activity, such as type 2 ribosome-inactivating proteins, might also pro...Chemically based rodent models are used to assess the positive effects promoted by foods and gut microbiota on gut health. Lectins with enzymatic activity, such as type 2 ribosome-inactivating proteins, might also prove useful for exploring these issues. Sub-lethal doses of the lectin nigrin from Sambucus nigra L. to mice promoted reversible derangement of gut epithelium by induction of apoptosis of transit amplifying cells of the small intestine crypts in a time-dependent course. The present work seeks to study vitamin B6 accumulation in plasma from an oral bolus in a mouse nigrin model. 24 h after sub-lethal nigrin b treatment, there was clear body weight reduction associated to a notable increase in Evan’s blue stain accumulation in excised small intestine, an increase in myeloperoxidase activity, and a near 50% reduction in plasma accumulation of vitamin B6. Histological analysis of small intestine sections of nigrin b-treated animals also revealed significant derangement of intestinal crypts. Seventy two hours after nigrin b treatment, stain uptake decreased and vitamin B6 accumulation was almost restored despite villi derangement. Large intestine crypts were scarcely or not at all affected. Eight days after nigrin b treatment, vitamin B6 uptake and intestinal crypt structure had fully recovered. The nigrin b mice model supports the view that, under these conditions, the carrier-mediated vitamin B6 uptake component of the small intestine crypts is probably the most active when the vitamin is administered orally as a bolus. The findings provide insights into the suitability of the present mice model for nutritional or drug absorption studies in conditions of partially altered or injured intestinal mucosa.展开更多
In the central nervous system,the A6 noradrenaline(NA)and the B3 serotonin(5-HT)cell groups are well-recognized players in the descending antinociceptive system,while other NA/5-HT cell groups are not well characteriz...In the central nervous system,the A6 noradrenaline(NA)and the B3 serotonin(5-HT)cell groups are well-recognized players in the descending antinociceptive system,while other NA/5-HT cell groups are not well characterized.A5/A7 NA and B25-HT cells project to the spinal horn and form descending pathways.We recorded G-Ca MP6 green fluorescence signal intensities in the A5/A7 NA and the B25-HT cell groups of awake mice in response to acute tail pinch stimuli,acute heat stimuli,and in the context of a non-noxious control test,using fiber photometry with a calcium imaging system.We first introduced G-Ca MP6 in the A5/A7 NA or B25-HT neuronal soma,using transgenic mice carrying the tetracycline-controlled transactivator transgene under the control of either a dopamineβ-hydroxylase or a tryptophan hydroxylase-2 promoters and by the site-specific injection of adeno-associated virus(AAV-Tet O(3 G)-G-Ca MP6).After confirming the specific expression patterns of G-Ca MP6,we recorded G-Ca MP6 green fluorescence signals in these sites in awake mice in response to acute nociceptive stimuli.G-Ca MP6 fluorescence intensity in the A5,A7,and B2 cell groups was rapidly increased in response to acute nociceptive stimuli and soon after,it returned to baseline fluorescence intensity.This was not observed in the non-noxious control test.The results indicate that acute nociceptive stimuli rapidly increase the activities of A5/A7 NA or B25-HT neurons but the non-noxious stimuli do not.The present study suggests that A5/A7 NA or B25-HT neurons play important roles in nociceptive processing in the central nervous system.We suggest that A5/A7/B2 neurons may be new therapeutic targets.All performed procedures were approved by the Institutional Animal Use Committee of Kagoshima University(MD17105)on February 22,2018.展开更多
基金Supported by National Foundation of Natural Sciences,China,No.81300293
文摘AIMTo investigate the protective effect of a recombinant adeno-associated virus carrying thymosin β<sub>4</sub> (AAV-Tβ<sub>4</sub>) on murine colitis via intracolonic administration.METHODSAAV-Tβ<sub>4</sub> was prepared and intracolonically used to mediate the secretory expression of Tβ<sub>4</sub> in mouse colons. Dextran sulfate sodium (DSS) was applied to induce the murine ulcerative colitis, and 2,4,6-trinitrobenzene sulfonic acid (TNBS) was used to establish a mouse colitis model resembling Crohn’s disease. The disease severity and colon injuries were observed and graded to reveal the effects of AAV-Tβ<sub>4</sub> on colitis. The activities of myeloperoxidase (MPO) and superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were determined using biochemical assays. Colonic levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-10 were measured using ELISA, and mucosal epithelial cell apoptosis and proliferation were detected by TUNEL assay and immunochemistry, respectively.RESULTSRecombinant AAVs efficiently delivered LacZ and Tβ<sub>4</sub> into the colonic tissues of the mice, and AAV-Tβ<sub>4</sub> led to a strong expression of Tβ<sub>4</sub> in mouse colons. In both the DSS and TNBS colitis models, AAV-Tβ<sub>4</sub>-treated mice displayed distinctly attenuated colon injuries and reduced apoptosis rate of colonic mucosal epithelia. AAV-Tβ<sub>4</sub> significantly reduced inflammatory cell infiltrations and relieved oxidative stress in the inflamed colons of the mice, as evidenced by decreases in MPO activity and MDA content and increases in SOD activity. AAV-Tβ<sub>4</sub> also modulated colonic TNF-α, IL-1β and IL-10 levels and suppressed the compensatory proliferation of colonic epithelial cells in DSS- and TNBS-treated mice.CONCLUSIONTβ<sub>4</sub> exerts a protective effect on murine colitis, indicating that AAV-Tβ<sub>4</sub> could potentially be developed into a promising agent for the therapy of inflammatory bowel diseases.
文摘Chemically based rodent models are used to assess the positive effects promoted by foods and gut microbiota on gut health. Lectins with enzymatic activity, such as type 2 ribosome-inactivating proteins, might also prove useful for exploring these issues. Sub-lethal doses of the lectin nigrin from Sambucus nigra L. to mice promoted reversible derangement of gut epithelium by induction of apoptosis of transit amplifying cells of the small intestine crypts in a time-dependent course. The present work seeks to study vitamin B6 accumulation in plasma from an oral bolus in a mouse nigrin model. 24 h after sub-lethal nigrin b treatment, there was clear body weight reduction associated to a notable increase in Evan’s blue stain accumulation in excised small intestine, an increase in myeloperoxidase activity, and a near 50% reduction in plasma accumulation of vitamin B6. Histological analysis of small intestine sections of nigrin b-treated animals also revealed significant derangement of intestinal crypts. Seventy two hours after nigrin b treatment, stain uptake decreased and vitamin B6 accumulation was almost restored despite villi derangement. Large intestine crypts were scarcely or not at all affected. Eight days after nigrin b treatment, vitamin B6 uptake and intestinal crypt structure had fully recovered. The nigrin b mice model supports the view that, under these conditions, the carrier-mediated vitamin B6 uptake component of the small intestine crypts is probably the most active when the vitamin is administered orally as a bolus. The findings provide insights into the suitability of the present mice model for nutritional or drug absorption studies in conditions of partially altered or injured intestinal mucosa.
基金supported by JSPS KAKENHI grants(Nos.19K17093 to SM20K06858 to AYamashita16H05130 to TK)and CREST JST(No.JPMJCR1656 to AYamanaka)。
文摘In the central nervous system,the A6 noradrenaline(NA)and the B3 serotonin(5-HT)cell groups are well-recognized players in the descending antinociceptive system,while other NA/5-HT cell groups are not well characterized.A5/A7 NA and B25-HT cells project to the spinal horn and form descending pathways.We recorded G-Ca MP6 green fluorescence signal intensities in the A5/A7 NA and the B25-HT cell groups of awake mice in response to acute tail pinch stimuli,acute heat stimuli,and in the context of a non-noxious control test,using fiber photometry with a calcium imaging system.We first introduced G-Ca MP6 in the A5/A7 NA or B25-HT neuronal soma,using transgenic mice carrying the tetracycline-controlled transactivator transgene under the control of either a dopamineβ-hydroxylase or a tryptophan hydroxylase-2 promoters and by the site-specific injection of adeno-associated virus(AAV-Tet O(3 G)-G-Ca MP6).After confirming the specific expression patterns of G-Ca MP6,we recorded G-Ca MP6 green fluorescence signals in these sites in awake mice in response to acute nociceptive stimuli.G-Ca MP6 fluorescence intensity in the A5,A7,and B2 cell groups was rapidly increased in response to acute nociceptive stimuli and soon after,it returned to baseline fluorescence intensity.This was not observed in the non-noxious control test.The results indicate that acute nociceptive stimuli rapidly increase the activities of A5/A7 NA or B25-HT neurons but the non-noxious stimuli do not.The present study suggests that A5/A7 NA or B25-HT neurons play important roles in nociceptive processing in the central nervous system.We suggest that A5/A7/B2 neurons may be new therapeutic targets.All performed procedures were approved by the Institutional Animal Use Committee of Kagoshima University(MD17105)on February 22,2018.
