Bavachin enhances NLRP3 inflammasome activation induced by ATP or nigericin and causes idiosyncratic hepatotoxicity / 医学前沿

Psoraleae Fructus (PF) is a well-known traditional herbal medicine in China, and it is widely used for osteoporosis, vitiligo, and other diseases in clinical settings. However, liver injury caused by PF and its preparations has been frequently reported in recent years. Our previous studies have demonstrated that PF could cause idiosyncratic drug-induced liver injury (IDILI), but the mechanism underlying its hepatotoxicity remains unclear. This paper reports that bavachin isolated from PF enhances the specific stimuli-induced activation of the NLRP3 inflammasome and leads to hepatotoxicity. Bavachin boosts the secretion of IL-1β and caspase-1 caused by ATP or nigericin but not those induced by poly(I:C), monosodium urate crystal, or intracellular lipopolysaccharide. Bavachin does not affect AIM2 or NLRC4 inflammasome activation. Mechanistically, bavachin specifically increases the production of nigericin-induced mitochondrial reactive oxygen species among the most important upstream events in the activation of the NLRP3 inflammasome. Bavachin increases the levels of aspartate transaminase and alanine aminotransferase in serum and hepatocyte injury accompanied by the secretion of IL-1β via a mouse model of lipopolysaccharide-mediated susceptibility to IDILI. These results suggest that bavachin specifically enhances the ATP- or nigericin-induced activation of the NLRP3 inflammasome. Bavachin also potentially contributes to PF-induced idiosyncratic hepatotoxicity. Moreover, bavachin and PF should be evaded among patients with diseases linked to the ATP- or nigericin-mediated activation of the NLRP3 inflammasome, which may be a dangerous factor for liver injury.

Cellular localization of NLRP3 inflammasome

Inflammasome is a large protein complex activated upon cellular stress or microbial infection, which triggers maturation of pro-inflammatory cytokines interleukin-1β and interleukin-18 through caspase-1 activation. Nod-like receptor family protein 3 (NLRP3) is the most characterized inflammasome activated by various stimuli. However, the mechanism of its activation is unclear and its exact cellular localization is still unknown. We examined the potential co-localization of NLRP3 inflammasome with mitochondria and seven other organelles under adenosine triphosphate, nigericin or monosodium urate stimulation in mouse peritoneal macrophages using confocal microscopy approach. Our results revealed that the activated endogenous apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome forms in the cytoplasm and co-localizes with NLRP3 and caspase-1, but not with any of the organelles screened. This study indicates that the ASC pyroptosome universally localizes within the cytoplasm rather than with any specific organelles.

Salinomicina e semduramicina associadas em diferentes concentrações no controle da eimeriose em frangos de corte / Salinomycin and semduramicin in different concentrations on the broilers eimeriosis control

O presente estudo teve por objetivo investigar a associação de salinomicina e semduramicina, em diferentes doses, frente à infecção mista controlada de Eimeria acervulina, E. maxima e E. tenella em frangos de corte. Oitocentas aves foram divididas em 5 grupos (T1: ração não medicada; T2: 30 ppm de salinomicina e 12,5 ppm de semduramicina; T3: 30 ppm de salinomicina e 15 ppm de semduramicina; T4: 40 ppm de salinomicina e 12,5 ppm de semduramicina e T5: 40 ppm de salinomicina e 15 ppm de semduramicina) e inoculadas aos 15 dias de idade com oocistos esporulados de E. acervulina, E. maxima e E. tenella, em inóculo misto, via ração. Parâmetros produtivos e escore de lesões foram registrados. Todos os grupos tratados apresentaram estatisticamente melhores ganhos de peso cumulativo aos 21 dias de vida. Aos 35 dias de vida, somente o grupo T3 apresentou diferença significativa. A conversão alimentar cumulativa apresentou diferença estatística nos grupos T4 e T5. O tratamento T5 foi mais eficaz no controle de E. tenella. T3 e T5 obtiveram diferenças estatísticas no escore médio de lesão das três espécies. O uso de salinomicina, associada a semduramicina, em baixas doses demonstrou uma opção viável no controle da coccidiose neste experimento.

This study aimed to investigate the association of salinomycin and semduramicin, in different doses, against controlled mixed infection of Eimeria acervulina, E. maxima and E. tenella in broiler chickens. Eight hundred birds were divided into 5 groups (T1: not medicated feed; T2: 30 ppm of salinomycin and 12.5 ppm of semduramicin; T3: 30 ppm of salinomycin and 15 ppm of semduramicin; T4: 40 ppm of salinomycin and 12,5 ppm of semduramicin and T5: 40 ppm of salinomycin and 15 ppm of semduramicin) and inoculated at 15 days of age with sporulated oocysts of E. acervulina, E. maxima and E. tenella in a mixed suspension, through the feed. Performance data and lesion scores were recorded. All treated groups showed statistically better cumulative weight gain at 21 days old. At 35 days old only the T3 group showed significant difference. Cumulative feed conversion showed statistical difference in the groups T4 and T5. The treatment T5 was more effective in the coccidiosis control of E. tenella. T3 and T5 achieved statistical differences in the average lesion scores of the three analyzed species. The association of salinomycin and semduramicin used in lower doses than the usual, showed to be an option in the coccidiosis control in this experiment.

Transfection and anti-HBV effect mediated by the hepatocytes-targeting cationic liposomes co-modified with beta-sitosterol-beta-D-glucoside and Brij 35 / 药学学报

<p><b>AIM</b>To study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.</p><p><b>METHODS</b>Dipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.</p><p><b>RESULTS</b>The co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.</p><p><b>CONCLUSION</b>Co-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.</p>

Metal ion specificity in anaesthetic induced increase in the rate of monensin and nigericin mediated H+/M+ exchange across phospholipid vesicular membranes.

From a study of the decay of the pH difference across vesicular membranes (delta pH) it has been possible to show that H+ and alkali metal ion (M+) concentration gradients across bilayer membranes (which are responsible for driving important biochemical processes) can be selectively perturbed by anaesthetics such as chloroform and benzyl alcohol by combining them with a suitable exchange ionophore. On adding the anaesthetic to the membrane in an environment containing metal ions M+ = K+, the rate of delta pH decay by H+/M+ exchange increases by a larger factor or by a smaller factor (when compared to that in a membrane environment with M+ = Na+) depending on whether the exchange ionophore chosen is monensin or nigericin. A rational explanation of this "metal ion specificity" can be given using the exchange ionophore mediated ion transport scheme in which the equilibrations at the "interfaces" are fast compared to the "translocation equilibration" between the species in the two layers of the membrane. The following three factors are responsible for the observed "specificity": On adding the anaesthetic (i) translocation rate constants increase, (ii) the concentrations of the M+ bound ionophores increase at the expense of H+ bound ionophores. (iii) Under our experimental conditions the rate determining species are the complexes monensin-K (Mon-K) and nigericin-H (Nig-H) for M+ = K+ whereas they are monensin-H (Mon-H) and nigericin-Na (Nig-Na) for M+ = Na+. Possible anaesthetic induced membrane perturbations contributing to the above mentioned changes in the membrane are (A), the loosening of the membrane structure and (B), an associated increase in the membrane hydration (and membrane dielectric constant). An analysis of the consequent changes in the various transport step shows the following: (a), The anaesthetic induced changes in the translocation rates of electrically charged species are not relevant in the explanation of the observed changes in the delta pH decay rates. (b), Changes in the rates of fast equilibria at the interface contribute to changes in KH and KM. (c), A suggestion made in the literature, that a significant interaction between the dipole moment of the monensin-K complex and the membrane slows down its translocation, is not valid. (d), The ability to explain rationally all the delta pH decay data confirms the validity of the transport scheme used. In our experiments delta pH across the vesicular membrane was created by pH jump coming from a temperature jump.

Swelling of the vesicle is prerequisite for PTH secretion

Unlike most secretory cells, high extra cellular calcium inhibits rather than stimulates hormonal secretion in several cells such as parathyroid cells, Juxtaglomerular cells and osteoclast. To gain further insight into the common but unique stimulus-secretion coupling mechanism in these cells, bovine parathyroid slices were incubated in various conditions of Krebs-Ringer (KR) solution containing essential amino acids. Parathyroid cells showed the inverse dependency of secretion on extra cellular calcium concentration as we expected. Ammonium acetate overcame the inhibitory effect of 2.5 mM of calcium and the maximum effect was as much as the five times of the basal value, while there was a little additive effect under 0 mM CaCl2. PTH secretion was biphasic according to the change of extra cellular osmolarity and the lowest response was observed at 300 mOsm/l. In Na-rich KR solution, high concentration of nigericin (> 10(-4)M) completely overcame the inhibitory effect of 2.5 mM CaCl2 and the maximum stimulatory effect was 8 times greater whereas it was only 2 times greater without CaCl2. In K-rich KR solution that abolished the K-gradient between the extra cellular solution and the cytoplasm, the rate of PTH secretion increased, and furthermore the addition of nigericin increased the rate of secretion significantly. The results above suggested that the osmotic swelling of the secretory vesicle in parathyroid cells might promote exocytosis as in Juxtaglomerular cells. We propose that the swelling of the vesicle is also prerequisite for secretion in several cells inhibited paradoxically by Ca++, whatever the signal transduction pathway for swelling of the secretory granules induced by the lowering of Ca++ in cytoplasm are.