Effect Of Artocarpus Lingnanensis Lectin On Growth Of B Cells And NK Cells And It's Mechanisms Involved

Objective:Artocarpus Lingnanensis lectin (ALL) is a kind of plant lectin that has been isolated and purified from the seeds of the Artocarpus lingnanensis, with stable properties and high agglutination activity. The previous study found that Artocarpus Lingnanensis Lectin(ALL) can effectively promote the proliferation and activation of human peripheral blood T lymphocytes, also can induce the mature and proliferation of human peripheral blood DC and BmDC. We also found that ALL could inhibit the proliferation of Jurkat T lymphocytes and EL-4 cells and induce them to apoptosis. In this research,NK92MI cells and B cells were used to explore the impact of ALL on their growth and its mechanisms involved. We hope to provides a theoretical and experimental foundation for further researches to the molecular mechanisms of ALL on clinical disease prevention and treatment.Methods:I. The effect of ALL on growth of B cells and NK92MI cells1. The effect of ALL on proliferation of B cellsMouse spleen B cell suspensions were prepared by Easy SepT Mouse B cell isolation kit under aseptic conditions. B cells were incubated with different concentration of ALL for 72 h, the proliferation of B cells was analyzed by CCK8 method and the change ofcell mor:phol ogy was observed by inverted microscope.2. The effect of ALL on proliferation of NK92MI cellsNK92MI cells were incubated with different concentration of ALL for 48 h,the proliferation of NK92MI cells was analyzed by CCK8 method and the change of cell morphology was observed by inverted microscope. The expression of granzyme A, granzyme B, granulysin and perforin were evaluatedby flow cytometry.?. The initial analysis of ALL receptorsThe binding of ALL to NK92MI cells was analyzed by FACS. The blocking of galactose and was analyzed by FACS. Flow cytometry was used to analyse the expression of ALL receptors and CD45. The colocalization of ALL receptors and CD45 were monitored under confocal microscope. The intemalizion of ALL by NK92MI cells was observed under confocal rmicroscope.?.The effect of ALL on apoptosis of NK92MI cells and its9 mechanisms involved.NK92MI cells were incubated with different concentration of ALL for 24h,the apoptosis of NK92MI cells was analyzed by FITC-Annexin V/PI staining;Apoptotic body in NK92MI cells was observed by transmission electron microscope. NK92MI cells synchronized by serum starvation were incubated with different concentrations of ALL, and the NK92MI cell cycle was analyzed by PI staining and FACS. The effect of galactose on ALL-induced apoptosis was analyzed by FITC-Annexin V/PI staining. The expression of apoptosis-related genes was analyzed by Real-Time PCR. The expression of caspase-3,cytochrome-c were evaluated by flow cytometry. The effect of caspase-3 inhibitor on ALL-induced apoptosis was analyzed by FITC-Annexin V/PI staining.Results:?. The effect of ALL on growth of B cells and NK92MI cells1. The effect of ALL on proliferation of B cellsAfter treatment by ALL for 72 h,B cells underwent morphological changes characteristic of apoptosis, such as membrane blebbing and cell shrinkage,whereas the cells did not show a change in morphology with no ALL treatment.CCK-8 assay showed that ALL could significantly inhibit the proliferation of B cells in a dose-dependent manner.2. The effect of ALL on proliferation of NK92MI cellsAfter treatment by ALL for 48 h, NK92MI cells underwent morphological changes characteristic of apoptosis, such as membrane blebbing and cell shrinkage, whereas the cells did not show a change in morphology with no ALL treatment. CCK-8 assay showed that ALL could significantly inhibit the proliferation of NK92MI cells in a dose-dependent manner. There was no significantly changes of granzyme A, granzyme B, granulysin, perforin expressing.?. The initial analysis of ALL receptorsThe addition of galactose exhibited significantly inhibition of the binding of ALL to NK92MI cells. Flow cytometry and confocal microscopy studies revealed that ALL exhibited binding to NK92MI cells and colocalized with CD45. For the further study, after treated with 13 ug/ml FITC-ALL for 30 min,60 min, 120 min , we found that ALL can be internalized by NK92MI cells.These cells showed labeling the membrane at 30 min of incubation with FITC-ALL. After 60 min, the NK92MI cells displayed intracellular labeling,maximum intracellular labeling was observed after 120 min of incubation.?. The effect of ALL on apoptosis of NK92MI cells and its' mechanisms involved.After treatment by ALL for 24 h, the apoptosis rate was incresased significantly compared to control cells. ALL induced apoptosis in a dose-dependent manner. Apoptotic body was observed clearly in ALL-treated cells by transmission electron microscope. NK92MI cells synchronized were incubated with ALL for 24h. The cell number in sub-G1 peak had increased. The addition of galactose exhibited significantly inhibition of protecting effect of ALL-induced apoptosis in NK92MI cells. The results of Real-Time PCR show that the expression of Bcl2?Bclxl?Bax was decreased in ALL-treated NK92MI cells. However, it has no significant difference of active caspase-3 and the intracellular levels of mitochondrial cytochrome-c between control andALL-treated NK92MI cells. Furthermore, the caspase-3 inhibitor,Z-DEVD-FMK, has no protecting effects on ALL-induced apoptosis in NK92MI cells.Conclusion:(1) ALL inhibited the proliferation of B cells and NK92MI cells.(2) CD45 may be one of ALL receptors on the surface of lymphocytes.(3 ) ALL may induce NK92MI cells apoptosis by entering the cells through reducing the expression of Bcl2 and Bclxl.