Study On The Toxicity Mechanism Of Araceae

Pinellia ternata (Thunb.) Breit., Pinellia pedatisecta Schott., Arisaema erubescens (Wall.) Schott., and Typhonium giganteum Engl., which belong to poisonous Chinese medicine. Three kinds of toxic drugs toxic irritant very similar, are expressed as hemp barbed tongue and throat. Swallowed all appear sore throat, difficulty breathing, severe cases death occurs. It is precisely because of this toxicity, clinical medicine to use their guns based products.Our previous have determined where Pinellia ternata (Thunb.) Breit., Pinellia pedatisecta Schott., Arisaema erubescens (Wall.) Schott. is a major irritant three toxic drugs toxic components, because of its special crystalline form, can produce severe irritation inflammation. Crystal can cause lethal rabbit conjunctival edema strong, enhanced capillary permeability in mice, rats paw swelling and other inflammatory reactions; preliminary results show a large number of elongated, flexible texture, and sharp pointed ends needle-like crystals can penetrate body tissue, causing pain and stimulate the inflammatory response, but the mechanism is not clear.Toxic raphides is mainly composed of calcium oxalate and proteins, but calcium oxalate has no irritating effect. Our previous research had proved that toxic raphides contain lectin, which Toxic raphides can further induce inflammation. The lectin can induce the migration of neutrophils to the sites of inflammation, which is an important step in cellular inflammatory responses, so the four lectin (PTL, PPL, AEL and TGL) can induce inflammation, but the mechanism of their action is not clear.Preliminary research of inflammatory irritation of PTL, PPL, AEL and TGL at the cellular level has been carry on. We found that these four lectins induced neutrophil migration is related to residence macrophages in vivo. Further, toxic raphides of P. ternate. and PTL stimulation on macrophages played defense, and induce significant release of pro-inflammatory factors, attracted neutrophils to inflammation sites, and activate NF-κB signal pathway. P. pedatisecta, A. erubescens and T. giganteum also from Araceae, toxic Substances foundation and irritation toxicity are similar to P. ternate. So, whether the toxic raphides and lectin of P. pedatisecta, A. erubescens and T. giganteum have proinflammatory effect? Whether the proinflammatory effect is related to macrophages? Therefore, this fourth chapter research the toxic raphides and lectin of P. pedatisecta, A. erubescens and T. giganteum. Put forward the hypothesis that the proinflammatory effect of P. pedatisecta, A. erubescens and T. giganteum closely related to macrophage mediation.The proinflammatory effect has its specific inflammatory mechanism, The findings herein provide valuable evidence for elucidating the toxic mechanism of Araceae. Therefore, this fifth chapter research the toxicity mechanism of the lectin of P. ternate, P. pedatisecta, A. erubescens and T. giganteum at the cellular level. Also study the impact of the four lectin on macrophage death patterns, and analyze its correlation with inflammation. Main work and results contain following parts:1 Study on the pro-inflammatory effect of toxic raphides of P. pedatisecta, A. erubescens and T. giganteumSEM was used to observed the morphology changes on the toxic raphides stimulated macrophages, the results show that after stimulation with 6.25μg/mL purified the three toxic raphides for 0.5 h and 2 h respectively, the morphology of macrophage did not change evidently. However, macrophage had increased volume and obvious wrinkles on the surface after being stimulated with 12.5 μg/mL the three toxic raphides for 2 h, suggesting that the macrophage was activated. After stimulation with 25 μg/mL the three toxic raphides for 0.5 h, pseudopod was produced on the cell surface, indicating that the macrophage was activated, and it died 2 h later owing to cytomembrane damages. Hence, macrophages involved in inflammation.Set up of macrophage culture model and study on the pro-inflammatory factors released from the three raphides stimulated macrophages, the results show that after stimulation with the three raphides at different concentrations for 1 h, TNF-a and IL-1β contents in the culture supernatants significantly exceeded those of the blank group, which increased dose-dependently. This result, together with SEM above, further verified that the proinflammatory effect of the three raphides is related to macrophages.Set up of macrophage-neutrophils co-culture model and neutrophils migration experiment was used to examine the relationship between the three toxic raphides stimulated macrophage and neutrophils migration, the results show that after the three toxic raphides (12.5μg/mL,25μg/mL,50μg/mL,100μg/mL) stimulation for 1 h, the number of migrating neutrophils increased compared with that of the PBS+Mφ group (P<0.01). Compared with the PBS group, neither the macrophage group nor the the three toxic raphides group significantly induced migration of neutrophils. Accordingly, in the macrophage-neutrophil co-culture system, the three toxic raphides stimulation dramatically induced the migration and aggregation of neutrophils through the mediation of macrophage. The migration of neutrophils was the first step of inflammatory response, so the proinflammatory effect of the three toxic raphides was closely related to macrophage.2 Study on the pro-inflammatory effect of PPL, AEL and TGLSEM was used to observed the morphology changes on PPL, AEL and TGL stimulated macrophages, the results show that after stimulation with 6.25μg/mL purified PPL, AEL and TGL for 0.5 h and 2 h respectively, the morphology of macrophage did not change evidently. However, macrophage had increased volume and obvious wrinkles on the surface after being stimulated with 12.5 μg/mL PPL, AEL and TGL for 2 h, suggesting that the macrophage was activated. After stimulation with 25 μg/mL PPL, AEL and TGL for 0.5 h, pseudopod was produced on the cell surface, indicating that the macrophage was activated, and it died 2 h later owing to cytomembrane damages. Hence, macrophages involved in inflammation.Set up of macrophage culture model and study on the pro-inflammatory factors released from PPL, AEL and TGL stimulated macrophages, the results show that after stimulation with PPL, AEL and TGL at different concentrations for 1 h, TNF-a and IL-1β contents in the culture supernatants significantly exceeded those of the blank group, which increased dose-dependently. This result, together with SEM above, further verified that the proinflammatory effect of PPL, AEL and TGL is related to macrophages.Set up of macrophage-neutrophils co-culture model and neutrophils migration experiment was used to examine the relationship between PPL, AEL and TGL stimulated macrophage and neutrophils migration. The results show that after the PPL, AEL and TGL (12.5μg/mL,25μg/mL,50 μg/mL,100μg/mL) stimulation for 1 h, the number of migrating neutrophils increased compared with that of the PBS+Mφ group (P<0.01). Compared with the PBS group, neither the macrophage group nor PPL, AEL and TGL group significantly induced migration of neutrophils. Accordingly, in the macrophage-neutrophil co-culture system, PPL, AEL and TGL stimulation dramatically induced the migration and aggregation of neutrophils through the mediation of macrophage. The migration of neutrophils was the first step of inflammatory response, so the proinflammatory effect of PPL, AEL and TGL was closely related to macrophage.3 Study on the pro-inflammatory mechanism of PPL, AEL and TGLUse Western Blot to evaluate the expression of NF-κB after PPL, AEL and TGL stimulation on macrophages. The results show that after stimulation with 12.5 μg/mL, 25 μg/mL and 50 μg/mL PPL, AEL and TGL for 1 h, the content of p65 in the macrophage cytoplasm plummeted compared with that of the blank group, but this content in the macrophage nucleus remarkably increased, all showing evident dose-effect relationships. Therefore, PPL, AEL and TGL stimulation caused transfer of p65 from the macrophage cytoplasm to the nucleus, which suggested the inflammation induced by PPL, AEL and TGL-stimulated macrophage was associated with the NF-κB signaling pathway.Macrophages were incubated with 1μM,5μM, 10μM,20μM of BAY 11-7082 for 1 h and then challenged with 50μg/mL PPL, AEL and TGL for 1 h. Compared with blank control, the content of p65 in cytoplasm was increased with higher BAY 11-7082 concentrations, while the nuclear fraction was significantly decreased proportionally. These results suggested that BAY 11-7082 can block nuclear translocation of p65 and effectively attenuate PPL, AEL and TGL induced macrophage activation. These findings may also establish a correlation between PPL, AEL and TGL induced macrophage activation and NF-kB signaling.4 Study death patterns of macrophageUse Flow Cytometry to study death patterns of macrophage stimulated by PPL, AEL and TGL. The results show that after stimulation with 50μg/mL PPL, AEL and TGL for 1h, a significant increase in the number of early apoptotic cells without necrosis, for 2-6h, necrosis began to appear, which increased time-dependently. The results showed that stimulation of PPL, AEL and TGL on macrophages eventually lead to macrophage necrosis.TEM was used to observe the internal structure of the morphological changes of macrophages by PPL, AEL and TGL after stimulation to determine their mode of death. The results show that 50μg/mL PPL, AEL and TGL stimulated macrophages 1～6h, compared with the control group, for 1h, nucleus chromatin margination, show that early apoptosis, without necrosis, for 2h, early apoptosis of macrophage cell membrane rupture few nuclei exposed, necrotic cells show the presence, for 4h, necrotic cells gradually increased, reached a maximum at 6h necrotic cells. This experiment is consistent with the above-described flow cytometry results confirmed PPL, AEL and TGL could induce macrophage inflammatory necrosis leading to generation.