ABSTRACT
Spinal cord injury (SCI) causes motor, sensory, and autonomic dysfunctions. The gut microbiome has an important role in SCI, while short-chain fatty acids (SCFAs) are one of the main bioactive mediators of microbiota. In the present study, we explored the effects of oral administration of exogenous SCFAs on the recovery of locomotor function and tissue repair in SCI. Allen's method was utilized to establish an SCI model in Sprague-Dawley (SD) rats. The animals received water containing a mixture of 150 mmol/L SCFAs after SCI. After 21 d of treatment, the Basso, Beattie, and Bresnahan (BBB) score increased, the regularity index improved, and the base of support (BOS) value declined. Spinal cord tissue inflammatory infiltration was alleviated, the spinal cord necrosis cavity was reduced, and the numbers of motor neurons and Nissl bodies were elevated. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (qPCR), and immunohistochemistry assay revealed that the expression of interleukin (IL)-10 increased and that of IL-17 decreased in the spinal cord. SCFAs promoted gut homeostasis, induced intestinal T cells to shift toward an anti-inflammatory phenotype, and promoted regulatory T (Treg) cells to secrete IL-10, affecting Treg cells and IL-17+ γδ T cells in the spinal cord. Furthermore, we observed that Treg cells migrated from the gut to the spinal cord region after SCI. The above findings confirm that SCFAs can regulate Treg cells in the gut and affect the balance of Treg and IL-17+ γδ T cells in the spinal cord, which inhibits the inflammatory response and promotes the motor function in SCI rats. Our findings suggest that there is a relationship among gut, spinal cord, and immune cells, and the "gut-spinal cord-immune" axis may be one of the mechanisms regulating neural repair after SCI.
Subject(s)
Animals , Rats , Interleukin-17 , Rats, Sprague-Dawley , Recovery of Function , Spinal Cord Injuries/drug therapy , T-Lymphocytes, Regulatory , Receptors, Antigen, T-Cell, gamma-delta/immunologyABSTRACT
Objective To construct the genetic recombinant adenovirus vector carrying the human growth and differentiation fac-tor-5(GDF-5) gene by using AdEasy-1 adenovirus vector system and to amplify and prepare the recombinant adenovirus in HEK 293 cells .Methods Human GDF-5 gene obtained by PCR was inserted into plasmid pMD19-T and the 1 .7 kb GDF-5 cDNA sequence was cloned into the adenoviral shuttle plasmid pShuttle-cytomegalovirus(CMV) of the AdEasy-1 system .It was identified by DNA sequencing and a digestion with Hind Ⅲ restriction enzyme .The resultant pShuttle-CMV-GDF-5 was used to generate the adenovi-ral GDF-5 vector through homologous recombination with the adenoviral backbone plasmid ,pAdEasy-1 in BJ5183 bacterial cells .It was selected by kanamycin and identified by a digestion with Hind Ⅲ restriction enzyme and amplified in XL10-Gold competent bac-teria .The DNA of recombinant adenovirus vector was finally linearized by Pac Ⅰ and the adenoviral recombinants were used to pro-duce adenoviruses in HEK293 packaging cells ,resulting in an Ad-GDF-5 identified by Western blot .The virus titer was assayed by TCID50 .Results GDF-5 cDNA sequence obtained by PCR was 1 .7 kb .Gene sequencing results indicated that the sequence was i-dentical with the one in GENBANK .Cloned sequence 1 .7 kb(GDF-5) was obtained by a digestion with Hind Ⅲ restriction enzyme after GDF-5 cDNA segment was cloned into pShuttle-CMV and AdEasy-1 .Western blot showed that two bands migrating at ap-proximately 15 and 55 kDa were clearly observed in PVDF membrane .These data confirmed that HEK293 cells expressed a large number of mature GDF-5 protein after infected with Ad-GDF-5 .Our research results demonstrated that recombinant adenovirus vector GDF-5 was successfully constructed .The virus titer was 5 .6 × 109 PFU/mL .Conclusion Recombinant adenovirus vector carrying the human GDF-5 gene is successfully constructed by using the AdEasy-1 adenovirus vector system .Moreover ,the Ad-GDF-5 with high titer is prepared .These provide the basis for further study of the biological function of GDF-5 and the gene thera-py of its related diseases .