Hydrogen sulfide preserves intestinal barrier repair function through sulfhydration of RPS20 in experimental colitis
Patients with ulcerative colitis (UC) have a significantly impaired intestinal barrier. Hydrogen Sulfide (H2S) is a gaseous mediator that makes notable contributions in a variety of diseases, such as reducing inflammatory response in colitis. The experimental content includes the establishment of a mouse DSS-induced colitis model, mouse colon epithelial organoids culture, H&E staining and mass spectrometry analysis. We recognized that exogenous H2S donor-GYY4137 significantly alleviated the symptoms in UC mice models and maintained Minichromosome Maintenance Complex Component 2 (MCM2) expression. CBS knockdown reduced the expression of sulfhydrated Ribosomal protein S20 (RPS20-ssh) and MCM2 in the mouse colon. Cell experiments indicated that the expression of RPS20-ssh, rather than total expression of RPS20, is responsible. Overall design: The CRISPR/Cas9 system was used to achieve stable knockout of the CBS gene in Caco-2 cells. In short, the interference target sgRNA was designed according to the sequence of the CBS gene as follows: CTGATGAGATCCTGCAGCAG. We phosphorylated, annealed, and cloned the guide oligonucleotide into the BsmBI site of the pHBLV-U6-gRNA-EF1-CAS9-PURO vector (Hanheng Biotechnology, China), and then verified the constructed vector by sequencing. The transferred plasmid with the guide oligonucleotide was then transformed into E. coli DH5a, and was isolated from the bacteria using a plasmid DNA purification kit (Tiangen, China). The transferable lenti-CAS-puro plasmid, packaging plasmids psPAX2 and pMD2G (Hanheng Biotechnology, China) were transfected into 293 T cells to produce lentivirus. 48 and 72 hours after transfection, the virus-containing supernatant was collected and then used to transfect Caco-2 cells. After 24 h of infection, the medium in each plate was replaced with fresh medium containing puromycin (30 mg/L) for 24 h, and then with fresh medium for 24 h. Three times of repetition were done. Finally, puromycin-resistant Caco-2 cells were collected and western blot was used to detect the CBS knockout efficiency.