The Separation, Purification And Identification Of HMGB1Secreted By Liver Cell Lines And Its Effects On Murine Macrophage

Objective:(1) To purify and identify high mobility group box-1(HMGB1) secreted by liver cell lines (HepG2).(2) To investigate the effects of HMGB1on the proliferation,the release of lactate dehydrogenase (LDH),apoptosis and the release of tumor necrosis factor-α (TNF-α), interleukin-1beta (IL-1β) and IL-6of RAW264.7cells.Methods:(1) The liver cell line (HepG2) and immune cell line (U937) were cultured and stimulated with HMGB1(400ng/mL), respectively. Twenty hours after stimulation, supernatant were collected and purified in turn with ultrafiltration centrifugation, CM-Sepharose cation, DEAE-Sepharose anion exchange chromatography, Sephadex G75-gel filtration chrom-atography, and immunoprecipita-tion. The purified HMGB1protein were identified by SDS-PAGE and Western blot.(2) After stimulated with different doses of HMGB1(10,50,100ng/mL) for24h or with100ng/mLHMGB1for different time points (8,24,48hours), the Cell proliferation of cultured RAW264.7cells was determined by MTT analysis and the supernatant of the cultured RAW264.7cells were collected to determine the level of LDH.(3) After stimulated with different doses of HMGB1(10,50,100ng/mL) for24hours or with100ng/mL HMGB1for different time points (8,24,48hours), the apoptosis of RAW264.7cells was determined by TUNEL assay.(4) After stimulated with different doses of HMGB1(10,50,100ng/mL) for24hours or with100ng/mL HMGB1for different time points(8,24,48 hours), the RAW264.7cells supernatants were harvested to determine the level of TNF-α IL-1β and IL-6by ELISA.Results:(1) A sharp stained protein band with a molecular weight of about26kD was obtained by SDS-PAGE analysis and confirmed to be HMGB1protein by Western-blotting.(2) Compared with no-treatment, different culture time and dosage of HMGB1displayed a profound inhibition effects on proliferation of RAW264.7cells (P<0.05).(3) Compared with no-treatment, different culture time and dosage of HMGB1displayed a profound promotion effects on release of LDH from RAW264.7cells (P<0.05).(4) Teatment of cultured RAW264.7cells with different dose of HMGB1(10,50,100ng/mL) for24hours resulted in a dose-dependent induction of apoptosis. Exposed to100ng/mLHMGB1for different time points (8,24,48hours), resulted in a time-dependent increase in apoptosis rate. There was significant difference between the treatment groups and the control group (P<0.05).The Percentage of apoptotic cells were markedly increased with a Peak value at48h after100ng/mL HMGB1treatment (P<0.05).(5) The expressions of TNF-α, IL-1β and IL-6in the supernatant were markedly up-regulated after stimulated with HMGB1for24hours. The effects of HMGB1on TNF-α, IL-1β and IL-6protein expressions displayed a dose-dependent manner, starting at dose as low as10ng/ml and showing a maximal response with100ng/ml(P<0.01). The effects of HMGB1on TNF-α,IL-1β and IL-6expressions were also time-dependent, with a significant increase within48hours (P<0.01).Conclusions:(1) Our purification methods are convenient, feasible and effective and the purity of separated HMGB1protein was relatively high.(2) HMGB1can significantly inhibited the proliferation of RAW264.7cells, promoted the releases of LDH in RAW264.7cells, induce the apoptosis of RAW264.7cells and promoted the release of TNF-α, IL-1β and IL-6in RAW264.7cells in a dose-and time dependent manner.