Influence of high mobility group box-1 protein inhibition on protein expression of rage/toll-like receptor 4 signal passway in ht22 cells induced with aβ25-35 / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To investigate the influence of high mobility group box-1 protein (HMGB1) inhibition on the expression of receptors for advanced glycation end products (RAGE)/Toll-like receptor 4 (TLR4) signal passway related proteins, RAGE, TLR4, NF-kB, interieukin-1β (IL-1βand tumor necrosis factor-α(TNF-α) in HT22 cells stimulated with amyloid-β25-35 (Aβ25-35). METHODS HT22 cells were stimulated with Aβ25-350, 2.5, 5, 10, 20 and 40 ymol • L"1 for 24 h, and the vitality of HT22 cells was determined using MTT assay. Half maximal inhibitory concentration (IC50) was calculated. HT22 cells were divided into 5 groups Normal cell control group, Apas-K 40 Mmol-L"' group, siRNA 50 Mmol-L"' group, siRNA or scramble siRNA+APas-as group (HT22 cells were transfected with siRNA or scramble siRNA at 50 pmol • L"' for 24 h, then treated with synthetic Apa-as at a final concentration of 40 pmol • L"1 for 24 h). The morphology of HT22 cells was observed under a microscope. The location of HMGB1 was observed by immunofluorescence. The protein expressions of HMGB1, RAGE, TLR4 and NF-kB P65 were detected by Western blotting. The levels of IL-IP and TNF-α in the culture supernatants were examined by ELISA. RESULTS The ICso of HT22 cells treated with Apas-as for 24 h was 41.17 Mmol-L-1, and A25-35 40 Mmol-L"1 was selected in subsequent experiments. After 24 h treatment with A25-35 40 nmol-L"', a large number of cells died, or aggregated into clusters, processes decreased, cell gaps increased, and HMGB1 was released from the nucleus to the outside. The protein expressions of HMGB1, RAGE, TLR4 and NF-kB P65 in HT22 cells were significantly increased (P<0.05) after treatment with Apas-as, and the levels of IL-1p and TNF-α in the culture supernatants were significantly increased (P<0.05). The protein expressions of HMGB1, RAGE, TLR4 and NF-kB P65 in HT22 cells were significantly decreased (P<0.05) after 24 h treatment with siRNA 50 pmol-L"' and the levels of IL-1p and TNF-α in the culture supernatants were significantly decreased (P<0.05). CONCLUSION RNA interference with HMGB1 reduces the expression of HMGB1, RAGE, TLR4 and NF-kB P65 in HT22 cells stimulated with A25-35