Recombinant adeno-associated virus carrying thymosin β4 suppresses experimental colitis in mice

Figure 1 Intracolonic administration of adeno-associated virus carrying thymosin β₄ mediated secretory expression of thymosin β₄ in mouse colons. Two weeks after intracolonic (ic) adeno-associated virus (AAV)-LacZ, AAV-thymosin β₄ (Tβ₄) or PBS, the mice were euthanized to harvest their colons. A: Real-time PCR showed the presence of viral DNA in the colonic tissues of the AAV-treated mice; B: X-gal staining verified AAV-mediated expression of LacZ in the colonic tissues. C: Immunohistochemistry revealed that ic AAV-Tβ₄ resulted in markedly increased expression of Tβ₄ in the mouse colon; D: In the mice with experimental colitis, Western blot showed that dextran sulfate sodium (DSS) treatment remarkably upregulated the colonic Tβ₄ expression while TNBS enemas only slightly increased colonic Tβ₄. Intracolonic AAV-Tβ₄ led to substantially increased expression of Tβ₄ in the colonic tissues in both DSS- and TNBS-treated mice. Error bars indicate SD. Scale bars = 50 μm.

Figure 2 Adeno-associated virus carrying thymosin β₄ ameliorated dextran sulfate sodium-induced colitis in mice. A: Dynamic changes in DAI [the asterisk indicates ^aP < 0.05 vs PBS/dextran sulfate sodium (DSS) or adeno-associated virus (AAV)-LacZ/DSS group]; B and C: AAV-thymosin β₄ (Tβ₄) improved DSS-induced colonic lesions including mucosal edema, hyperemia, and ulceration, resulting in a significantly decreased macroscopic score; D and E: Histological examination (HE staining, magnification × 200) revealed the mice in AAV-Tβ₄/DSS group exhibited alleviated colonic lesions such as destruction of mucosal crypts, loss of epithelial cells, deep ulcerations, and inflammatory cell infiltration, leading to a significantly decreased histological score compared with the mice in the other two DSS groups. Error bars indicate the SD. Scale bars = 50 μm. DAI: Disease activity index.

Figure 3 Adeno-associated virus carrying thymosin β₄ improved 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. A: Dynamic changes in DAI [the asterisk indicates ^aP < 0.05 vs PBS/2,4,6-trinitrobenzene sulfonic acid (TNBS) or adeno-associated virus (AAV)-LacZ/TNBS group]; B: TNBS treatment resulted in hyperemia, edema and ulceration, which were attenuated by Tβ₄; C: Macroscopic score of AAV-Tβ₄/TNBS group was significantly lower than those of the other two TNBS groups; D and E: Histological observation of colonic tissue (HE staining, magnification × 200) showed that AAV-Tβ₄ relieved TNBS-induced colonic mucosal lesions and reduced the histological score. Error bars indicate the SD. Scale bars = 50 μm. DAI: Disease activity index.

Figure 4 Adeno-associated virus carrying thymosin β₄ suppressed dextran sulfate sodium - and 2,4,6-trinitrobenzene sulfonic acid-induced inflammation in colonic tissue, as indicated by myeloperoxidase activity assessment. Error bars indicate the SD. AAV: Adeno-associated virus; Tβ₄: Thymosin β₄; DSS: Dextran sulfate sodium; TNBS: 2,4,6-trinitrobenzene sulfonic acid.

Figure 5 Adeno-associated virus carrying thymosin β₄ alleviated dextran sulfate sodium- and 2,4,6-trinitrobenzene sulfonic acid-induced epithelial cell apoptosis in the colonic mucosa. TUNEL staining showed that intracolonic adeno-associated virus (AAV)-thymosin β₄ (Tβ₄) prevented the colonic mucosal epithelia from undergoing apoptosis and thus led to decreased apoptotic indexes (AIs) in dextran sulfate sodium (DSS)- (A and B) and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced (C and D) colitis in mice. The insets indicate the TUNEL-positive nuclear staining. Error bars indicate the SD. Scale bar: 50 μm and 25 μm (insets).

Figure 6 Adeno-associated virus carrying thymosin β₄ relieved colonic oxidant damage in the mice with colitis. MDA content (A) and SOD activity (B) were determined using biochemical assays. Error bars indicate the SD. AAV: Adeno-associated virus; Tβ₄: Thymosin β₄.

Figure 7 Adeno-associated virus carrying thymosin β₄ modulated colonic TNF-α, IL-1β and IL-10 levels in dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid models. Colonic TNF-α (A), IL-1β (B) and IL-10 (C) levels were determined by ELISA. Error bars indicate the SD. AAV: Adeno-associated virus; Tβ₄: Thymosin β₄; DSS: Dextran sulfate sodium; TNBS: 2,4,6-trinitrobenzene sulfonic acid.

Figure 8 Adeno-associated virus carrying thymosin β₄ attenuated the acceleration of colonic mucosal cell proliferation following dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid treatments. Colonic cell proliferation was determined by PCNA immunostaining in dextran sulfate sodium (DSS) (A) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) models (C). PCNA label indexes (PCNA LIs) of the DSS model (B) and TNBS model (D) were calculated. Error bars indicate the SD. Scale bars = 50 μm. AAV: Adeno-associated virus; Tβ₄: Thymosin β₄.