P390 New features of molecular diagnostics of the regulation of molecular apoptotic pathway in ulcerative colitis
A.A. Iakovlev, A. Volkov, I. Shcherbakova, G. Tarasova, S. Malakhanov, O. Bashtovaya
Department of Gastroenterology, Rostov State Medical University, Rostov-on-Don, Russian Federation
Background
The incidence rate of ulcerative colitis (UC) in Russia is 5–30 cases per 100,000 per year. Molecular pathways of the UC development are not clear. The purpose of the work was to study the molecular interactions of the apoptotic pathway in patients with UC.
Methods
The clinical trial included 92 patients with UC. The clinical presentation of UC depends on the extent of involvement: distal colitis, extensive colitis; the severity of the disease. Criteria of the diagnosis of UC corresponded to ECCO Consensus. Mucosal specimens were graded according to grades 0, 1, 2, 3. The separation of proteins of colon mucosa was based on technologies of IEF, SDS-PAGE, 2DPAGE, by standard sets (MB-HIC C8 Kit, MB-IMAC Cu, MB-Wax Kit, Bruker, USA). The getting of mass-spectrogram was determined by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry (MALDI-TOF-MS/MS, Ultraflex II, Bruker, USA). Statistical analysis was performed using the software Statistica 10.0 (Statsoft).
Results
HSP2 controls the apoptosis of colonocytes and immune response in damaged colon mucosa by Bcl-2 and IL-17, and is also responsible for the resistance to therapeutic strategies. Anti-apoptotic functions of HSP27 is possible through the interaction with DAXX7, the activation of Akt and the inhibition of the apoptosis. HSP47 interacts with collagen I, II, III, IV and V types, that contributes to the launch of autoimmune process in UC. Caspase 8 protects colonocytes from TNFα-induced cell death through necroptosis mechanism via the blockade of the expression of RIP3. Significant decrease of the expression of PPARγ promotes the activation of STAT and AP-1 signalling pathways, that promotes the increase of the synthesis of IL-2, -6, -8, -12, TNFα, matrix metalloproteinases, the activity of immune and inflammation processes in the colon mucosa.
Results of qualitative proteomic analysis in patients with UC (Tab.)
| Protein name | Mw, Da | pI | Patients with active UC, n | Patients with the UC remission, n | Patients with relapses, n | ||
| TNF- α | 25,644 | 6.4 | 31 | 11 | 6.14 | 28 | 4.98 |
| NF-kB | 105,356 | 5.5 | 7 | 25 | 4.77 | 3 | 6.18 |
| Cytochrome C | 11,749 | 9.6 | 30 | 4 | 7.66 | 30 | 7.66 |
| Apaf-1 | 141,840 | 5.9 | 30 | 4 | 7.66 | 23 | 5.20 |
| Caspase 8 | 55,391 | 5.1 | 31 | 5 | 8.00 | 25 | 5.42 |
| Caspase 9 | 46,281 | 5.7 | 30 | 3 | 7.95 | 25 | 6.18 |
| Caspase 10 | 58,951 | 5.9 | 29 | 5 | 6.82 | 23 | 4.84 |
Conclusion
Proteomic analysis has demonstrated the cascade with caspases 8, 9, 10, the release of cytochrome C from mitochondria, which interacts with Apaf-1, causing activation of caspase-9 in UC colon mucosa. TNF-α activates of initiator caspase-2, -8, and -10 in the apoptotic pathway. The NF-kB pathway induces cellular inhibitors of apoptosis, which function as specific caspase inhibitors in UC colonic mucosa.