Novel syngeneic liver hapten protein compounds play different roles in pathogenesis of autoimmune hepatitis in the C57BL/6 mouse.

BACKGROUND/AIMS: The autoimmune hepatitis (AIH) model in C57BL/6 mice with syngeneic hapten S100 and adjuvant injected intraperitoneally has been designed to elucidate the pathogenesis of AIH. Three separate hapten peak proteins, peak I, peak II and peak III, could be derived from S100, but little is understood their roles on the development of AIH. This study aims to learn more about these roles on pathogenesis of AIH. METHODOLOGY: Novel AIH C57BL/6 mouse models were developed by weekly immunization by intraperitoneal injection with syngeneic S100 liver proteins and the three separated hapten peak proteins emulsified covalently in complete Freund's adjuvant (CFA) for 4 weeks and sacrificed for liver histopathological study. Additionally, TNF-α and INF-γ in culture supernatants of spleen lymphocytes of healthy C57BL/6 mice cultured together with S100 plus CFA for 48 hours were detected, and the T-lymphocytes proliferative response after stimulation with crude S100, peak I, II or III proteins were also assessed. RESULTS: Data showed that hepatitis induced by CFA+S100 was accompanied with more severe inflammation characterized by diffusely distributed liver necrosis and enhanced lymphocyte infiltration in portal tracts, while hepatitis induced by peak I+CFA was characterized by mass lymphocyte infiltration, occasional isolated liver necrosis and many acidophilic bodies, which was more similar to autoimmune hepatitis; hepatitis induced by peak II+CFA was characterized by massive liver necrosis and mild lymphocyte infiltration; hepatitis induced by peak III+CFA was characterized by mild inflammation with isolated acidophilic bodies or dotted hepatocellular necrosis. TNF-α, INF-γ from culture supernatants were increased, and T-lymphocyte proliferative response stimulated with peak I protein significantly increased compared with those stimulated with crude S100, peak II or III proteins. CONCLUSIONS: Syngenic S100 liver protein and its three separated hapten proteins have different roles in the pathogenesis of AIH, and peak I protein may be important in its development.

Effects of magnetic nanoparticle of Fe3O4 on apoptosis induced by Gambogic acid in U937 leukemia cells / 中国实验血液学杂志

This study was aimed to explore the potential therapy of Gambogic acid (GA) combined with magnetic nanoparticle of Fe3O4 (Fe3O4-MNP) on leukemia. The proliferation of U937 cells and the cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscopy and flow cytometry respectively. The expressions of gene and protein were detected by quantitative real-time polymerase chain reaction and Western blot respectively. The results showed that GA enhanced the cytotoxicity for U937 cells in dose- and time-dependent manners. The Fe3O4-MNP itself had not cytotoxicity, but could enhance the inhibitory effect of GA on proliferation of U937 cells. The apoptotic rate of U937 cells induced by combination of GA with Fe3O4-MNP was higher than that by GA alone. The typical apoptotic features of cells treated with GA and Fe3O4-MNP were observed. The expression levels of caspase-3 and bax after co-treatment of GA and Fe3O4-MNP were higher than that exposed to GA or Fe3O4-MNP alone, but the expressions of bcl-2, NF-kappaB and survivin were down-regulated. It is concluded that Fe3O4-MNP can promote GA-induced apoptosis in U937 cells, and the combination of GA with Fe3O4-MNP may be a safer and less toxic new therapy for leukemia.

Effects of magnetic nanoparticle of fe(3)o(4) and 5-bromotetrandrine on apoptosis of K562/A02 leukemic cells induced by daunorubicin / 中国实验血液学杂志

The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe(3)O(4) and 5-Bromotetrandrine (5-BrTet) on chronic leukemia. The apoptosis was detected by flow cytometry (FCM), Wright staining and light microscope; the expressions of BAX and BCL-2 were measured by Western blot. The results showed that combination of daunorubicin (DNR) with either MNP (Fe(3)O(4)) or 5-BrTet exerted a potent cytotoxic effect on K562/A02 cells, while MNP (Fe(3)O(4)) and 5-BrTet co-treatment could synergistically enhance DNR-induced apoptosis. After treated with this regimen, the typical apoptotic morphological features were found in K562/A02 cells; the expression level of BCL-2 decreased and BAX increased markedly. It is concluded that MNP (Fe(3)O(4)) or 5-BrTet with DNR can induce apoptosis in K562/A02 cells, and they show distinct synergism when used together. The down-regulation of BCL-2 and the up-regulation of BAX may play important roles.

Reversal of multidrug resistance in xenograft nude-mice by magnetic Fe(3)O(4) nanoparticles combined with daunorubicin and 5-bromotetrandrine / 中国实验血液学杂志

This study was aimed to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe(3)O(4) (Fe(3)O(4)-MNPs) combined with DNR in vivo. The xenograft leukemia model with stable multiple drug resistance in nude mice was established. The two sub-clones of K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1 x 10(7) cells/each) to establish the leukemia xenograft models. Drug resistant and the sensitive tumor-bearing nude mice were both assigned randomly into 5 groups: group A was treated with NS; group B was treated with DNR; group C was treated with nanoparticle of Fe(3)O(4) combined with DNR; group D was treated with 5-BrTet combined with DNR; group E was treated with 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were carried out. The protein levels of BCL-2, BAX, and Caspase-3 in resistant tumors were detected by Western blot. The results indicated that 5-BrTet and magnetic nanoparticle of Fe(3)O(4) combined with DNR significantly suppressed growth of K562/A02 cell xenograft tumor, histopathologic examination of tumors showed the tumors necrosis obviously. Application of 5-BrTet and magnetic nanoparticle of Fe(3)O(4) inhibited the expression of BCL-2 protein and up-regulated the expression of BAX, and Caspase-3 protein in K562/A02 cell xenograft tumor. It is concluded that 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR have significant tumor-suppressing effect on MDR leukemia cell xenograft model.

Daunorubicin-loaded magnetic nanoparticles of Fe(3)O(4) greatly enhance the responses of multidrug-resistant K562 leukemic cells in a nude mouse xenograft model to chemotherapy / 中国实验血液学杂志

Multidrug resistance (MDR) plays a major role in the failure of cancer chemotherapy. Since Fe(3)O(4)-magnetic nanoparticle loaded with daunorubicin (DNR) can overcome multidrug-resistance of K562 cells in vitro, the effect of Fe(3)O(4)-magnetic nanoparticle loaded with DNR on multidrug-resistant K562 cells was studied in vivo, the K562-n and its MDR counterpart K562-n/VCR cells were inoculated subcutaneously into both sides of the back of nude mice to establish a human leukemia xenograft model. The mice were randomly divided into group A receiving normal saline, group B receiving DNR, group C receiving Fe(3)O(4)-magnetic nanoparticle, group D receiving Fe(3)O(4)-magnetic nanoparticle loaded with DNR and group E receiving Fe(3)O(4)-magnetic nanoparticle containing DNR with a magnetic field built on the surface of the tumor tissue. The tumor volume was measured on the day 1, 5, 9, 13, 17 and 21 after the first treatment. Tumor tissues were isolated for examination of the expression of mdr-1 by reverse transcription polymerase chain reaction and Western blotting. The results showed that for K562-n/VCR tumor, the tumor volume was markedly lower in groups D and E than that in groups A, B and C. Pathological observation revealed that the tumor cells of group A and B grew well, some disseminated necrosis and some cells with karyorrhexis and karyopyknosis existed in group C. However, significant fracture, necrosis of cell and subsequently fibrosis were seen in group D and E. The transcription of mdr-1 gene in groups D and E was significantly lower than that in groups A, B and C (group D and E vs group A, B or C, p < 0.05). However, there were no differences about the protein expression of P-gp between these groups. The tumor volume of K562-n in groups C, D and E was markedly lower than that in groups A and B (group C, D and E vs group A or B, p < 0.05). Pathological observation showed that the tumor cell of group A and B grew well, and no obvious necrosis was observed. Significant fracture, necrosis of cell and subsequently fibrosis were seen in group C, D and E. It is concluded that DNR-loaded Fe(3)O(4) magnetic nanoparticles can suppress the growth of the MDR K562-n/VCR tumor in vivo, but can not further enhance its efficacy on the sensitive K562-n tumor as compared to DNR alone. The additional external magnetic field failed to further improve the antitumor effect in vivo.