Mechanism of dexmedetomidine protection against cisplatin induced acute kidney injury in rats.

OBJECTIVE: This study explored the molecular mechanisms by which dexmedetomidine (Dex) alleviates cisplatin (CP)-induced acute kidney injury (AKI) in rats. METHODS: CP-induced AKI models were established, and Dex was intraperitoneally injected at different concentrations into rats in the model groups. Subsequently, rats were assigned to the control, CP, CP + Dex 10 µg/kg, and CP + Dex 25 µg/kg groups. After weighing the kidneys of the rats, the kidney arterial resistive index was calculated, and CP-induced AKI was evaluated. In addition, four serum biochemical indices were measured: histopathological damage in rat kidneys was detected; levels of inflammatory factors, interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor alpha, in kidney tissue homogenate of rats were assessed through enzyme-linked immunosorbent assay (ELISA); and levels of NLRP-3, caspase-1, cleaved caspase-1, gasdermin D (GSDMD), and GSDMD-N in kidney tissues of rats were determined via western blotting. RESULTS: Dex treatment reduced nephromegaly and serum clinical marker upregulation caused by CP-induced AKI. In addition, hematoxylin and eosin staining revealed that Dex treatment relieved CP-induced kidney tissue injury in AKI rats. ELISA analyses demonstrated that Dex treatment reduced the upregulated levels of proinflammatory cytokines in the kidney tissue of AKI rats induced by CP, thereby alleviating kidney tissue injury. Western blotting indicated that Dex alleviated CP-induced AKI by inhibiting pyroptosis mediated by NLRP-3 and caspase-1. CONCLUSION: Dex protected rats from CP-induced AKI, and the mechanism may be related to NLRP-3/Caspase-1-mediated pyroptosis.

Bhlhe40 deficiency attenuates LPS-induced acute lung injury through preventing macrophage pyroptosis.

BACKGROUND: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) as common life-threatening lung diseases with high mortality rates are mostly associated with acute and severe inflammation in lungs. Recently, increasing evidence supports activated inflammation and gasdermin D (GSDMD)-mediated pyroptosis in macrophage are closely associated with ALI. Basic helix-loop-helix family member e40 (Bhlhe40) is a transcription factor that is comprehensively involved in inflammation. However, there is little experimental evidence connecting Bhlhe40 and GSDMD-driven pyroptosis. The study sought to verify the hypothesis that Bhlhe40 is required for GSDMD-mediated pyroptosis in lipopolysaccharide (LPS)-induced inflammatory injury. METHOD: We performed studies using Bhlhe40-knockout (Bhlhe40 -/-) mice, small interfering RNA (siRNA) targeting Bhlhe40 and pyroptosis inhibitor disulfiram to investigate the potential roles of Bhlhe40 on LPS-induced ALI and the underlying mechanisms. RESULTS: Bhlhe40 was highly expressed in total lung tissues and macrophages of LPS-induced mice. Bhlhe40-/- mice showed alleviative lung pathological injury and inflammatory response upon LPS stimulation. Meanwhile, we found that Bhlhe40 deficiency significantly suppressed GSDMD-mediated pyroptosis in macrophage in vivo and in vitro. By further mechanistic analysis, we demonstrated that Bhlhe40 deficiency inhibited GSDMD-mediated pyroptosis and subsequent ALI by repressing canonical (caspase-1-mediated) and non-canonical (caspase-11-mediated) signaling pathways in vivo and in vitro. CONCLUSION: These results indicate Bhlhe40 is required for LPS-induced ALI. Bhlhe40 deficiency can inhibit GSDMD-mediated pyroptosis and therefore alleviate ALI. Targeting Bhlhe40 may be a potential therapeutic strategy for LPS-induced ALI.

The SMAC mimetic birinapant alleviates lipopolysaccharide-induced acute lung injury by inhibiting MAPK signaling.

The second mitochondria-derived activator of caspases (SMAC) mimetic birinapant attenuated liver injury by inhibited the degradation of tumor necrosis factor receptor-associated factor 3 (TRAF3) and activation of mitogen-activated protein kinase (MAPK) signaling pathway in liver macrophage, but its role in LPS induced acute lung injury (ALI) is not understood. The present study was to investigate the effects of birinapant on ALI and its possible mechanism. A dose of birinapant (30 mg/kg) or a vehicle was administered intravenously 24 hours before LPS (100 µg) stimulation in mice. The levels of TNF-α, IL-6 and IL-1ß in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The infiltrated macrophages and expression of monocyte chemoattractant protein-1 (MCP-1) was determined by immunohistochemistry staining in the lung tissues. The JNK and p38 MAPK activation, protein expression and K48-linked polyubiquitination of TRAF3 were determined in alveolar macrophage cell line (MH-S cells) after 1µg/ml LPS stimulation. The results showed that the birinapant down-regulated the levels of TNF-α, IL-6 and IL-1ß in the BALF. In addition, birinapant markedly inhibited macrophages infiltration and MCP-1 protein expression in lung tissues. At last, birinapant suppressed the MAPKsignaling pathway and K48-linked ubiquitinated degradation of TRAF3 in MH-S cells after LPS administration. In conclusion, the results proved that birinapant protected against LPS-induced ALI through inhibiting MAPK activation and K48-linked ubiquitination of TRAF3 in alveolar macrophages.

Atorvastatin reduces contrast media-induced pyroptosis of renal tubular epithelial cells by inhibiting the TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND: Contrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure. However, there is no effective treatment of CI-AKI, and its mechanism is unknown. Interestingly, atorvastatin has been reported to be effective in renal injury. Therefore, the aim of this study was to explore the effect and possible molecular mechanism of atorvastatin in CI-AKI. METHODS: On the CI-AKI in vitro model, rat tubular epithelial cells (NRK-52E) were treated with 18 mg I/ml meglumine diatrizoate (MEG) and then pretreated with atorvastatin. pcDNA3.1-TLR4 treatment was performed to overexpress toll-like receptor 4 (TLR4) in NRK-52E cells. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) kits were used to detect NRK-52E cell viability as well as LDH release in each group, respectively; qRT-PCR to determine mRNA expression of TLR4 in cells; western blot to detect protein expression levels of pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD) and TLR4/MyD88/NF-κB signaling pathway-related proteins (TLR4, MyD88, NF-κBp65, and p-NF-κB p65) in cells. RESULTS: MEG treatment significantly inhibited the viability of NRK-52E cells, increased pro-inflammatory factor levels and promoted pyroptosis, representing successful establishment of a rat tubular epithelial cell (NRK-52E) CI-AKI in vitro model. Notably, atorvastatin increased the activity of MEG-treated NRK-52E cells and alleviated cell injury in a concentration-dependent manner. In addition, atorvastatin significantly down-regulated the expression of TLR4 in MEG-treated NRK-52E cells. However, overexpression of TLR4 inhibited the effects of atorvastatin on increasing cell viability, alleviating cell injury, reducing pro-inflammatory factors (IL-1ß, IL-6, and TNF-α) levels, and inhibiting apoptosis (by down-regulating the expression of NLRP3, caspase-1, ASC, and GSDMD). Furthermore, atorvastatin also inhibited the expression of TLR4/MyD88/NF-κB pathway-related proteins (TLR4, MyD88, and p-NF-κB p65). CONCLUSION: Atorvastatin can attenuate CI-AKI through increasing the activity of MEG-treated renal tubular epithelial cells, relieving cell injury, as well as inhibiting pyroptosis and inflammation. More importantly, the mechanism was achieved by inhibiting the TLR4//MyD88/NF-κB signaling pathway.

Inhibition of Caspase-11-Mediated Pyroptosis Alleviates Acute Kidney Injury Associated with Severe Acute Pancreatitis in Rats.

Background: Acute kidney injury (AKI) is a common complication in patients with severe acute pancreatitis (SAP). Caspase-11-mediated pyroptosis is essential for the progression of multiple diseases, but its role in SAP-induced AKI remains unknown.Aims: This research investigated whether caspase-11-mediated pyroptosis is involved in SAP-induced AKI and whether inhibiting caspase-11-mediated pyroptosis improves SAP-induced AKI.Methods: A rat model of SAP with AKI was established by slowly injecting 5% sodium taurocholate into the biliopancreatic duct, then wedelolactone (25 or 50 mg/kg), an inhibitor of caspase-11, was injected through the intra-peritoneum 1 and 6 h after SAP induction. Serum biochemical indexes, including serum amylase, lipase, interleukin (IL)-6, blood urea nitrogen (BUN), tumor necrosis factor (TNF)-α, and creatinine (Cr) in rats, were evaluated using biochemical test kits. Caspase-11 and gasdermin D (GSDMD) expression in the kidney tissues was evaluated by western blotting and immunohistochemical staining. IL-1ß and IL-18 levels in kidney tissues were detected by ELISA kits. Furthermore, histopathological alterations of pancreas and kidney were assessed by H&E staining.Results: The serum biochemical indexes and pyroptosis-related proteins in kidney tissues were significantly increased after SAP induction. Furthermore, wedelolactone decreased the expression of pyroptosis-linked proteins in kidney tissues, reduced serum lipase, amylase, IL-6, TNF-α, BUN, and Cr, and ameliorated the renal and pancreatic histological damage in SAP rats.Conclusion: Caspase-11-mediated pyroptosis contributes to SAP-induced AKI, and targeting caspase-11-mediated pyroptosis might be a novel treatment strategy for SAP-induced AKI.

Apoptose e expressão de Bcl-2 e das caspases 3 e 8 em placenta bovina, em diferentes estádios de gestação / Apoptosis and expression of Bcl-2 and caspases 3 and 8 in placenta of cows at different pregnancy stages

Apoptose e seus mecanismos reguladores são eventos fisiológicos cruciais para a manutenção da homeostase placentária, e o desequilíbrio desses processos pode comprometer a função placentária e, consequentemente, o sucesso da gravidez. Neste estudo, investigou-se a apoptose utilizando-se histomorfometria em lâminas coradas em HE e submetidas à reação de TUNEL. Além disso, avaliou-se a expressão de Bcl-2 e das caspases 8 e 3, pela reação de polimerase em cadeia em tempo real, em placentas saudáveis em diferentes estádios de gestação. Amostras de placentônios de vacas com quatro, seis e nove meses de gestação foram colhidas e processadas. O índice apoptótico aumentou progressivamente com o avanço da gestação. Tanto o Bcl-2 quanto as caspases 3 e 8 foram expressas nos três períodos estudados, sendo a expressão de Bcl-2 menor que a de caspase 8, que é menor que a de caspase 3. Estes resultados indicam que essas moléculas estão envolvidas na via apoptótica ativada na maturação placentária, exibindo um padrão de expressão ao longo da gestação e contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.

Apoptosis and its regulating mechanisms are crucial physiological events for the maintenance of the placental homostasis; and disequilibrium of these processes may compromise placental function and the success of the pregnancy. In this study, apoptosis was investigated by histomorphometry using slides stained with HE and TUNEL reaction. Besides that, Bcl-2 and caspases 8 and 3 expression were evaluated by real time polymerase chain reaction in healthy placentas under different gestacional ages. Samples of placentones of cows at 4th, 6th, and 9th months of gestation were harvested and processed. The apoptotic index gradually increased with the advance of the gestation. Bcl-2 and caspases 3 and 8 were expressed in all the studied periods, being the expression of Bcl-2 lower than that of caspase 8, which was lower than caspase 3. These results indicate that these molecules are involved in the activated apoptotic way in the placental maturation, showing a standard expression throughout the gestation and contributing for the physiological balance of the cellularity and cellular turn over in bovine placenta.

Expressão gênica de caspases 3 e 8 em timo e baço de ratas recém-desmamadas e imunossuprimidas por glicocorticóide / Genic expression of caspases 3 and 8 in thymus and spleen of glicocorticoid-immunossupressed weaned rats

Determinou-se a expressão gênica das caspases 3 e 8 mediante transcrição reversa de mRNA total e reação em cadeia da polimerase (RT-PCR) para avaliar a apoptose em timo e baço de ratas imunossuprimidas por glicocorticóides. Utilizou-se dexametasona para indução da apoptose e atrofia linfóide. Quarenta e cinco fêmeas Wistar recém-desmamadas foram separadas em três grupos: as ratas de A (n=18) e B (n=18) foram tratadas com 250 e 500mg de glicocorticóide, via intramuscular, respectivamente, e as do C (n=9) não foram tratadas. Após 24, 48 e 72 horas, seis animais de cada grupo tratado e três do controle foram anestesiados, pesados e sacrificados. O baço e o timo foram coletados e pesados. Fragmentos dos órgãos foram fixados em formol tamponado a 10 por cento e processados segundo técnica para inclusão em parafina. Os blocos foram seccionados em 5æm, e os cortes corados em hematoxilina e eosina. A análise histopatológica aliada ao peso dos órgãos nas diferentes doses e tempos demonstrou que a dexametasona induziu hipotrofia linfóide, que ocorreu com maior intensidade no tempo de 72 horas em animais do grupo B. Fragmentos de timo e de baço foram imediatamente congelados em nitrogênio líquido para extração de mRNA e DNA. Para a padronização da técnica de RT-PCR, utilizaram-se pool de amostras de mRNA dos animais-controle e pool de mRNA de animais tratados em cada tempo de experimento. A técnica de RT-PCR foi sensível o suficiente para a detecção dos mRNAs que codificam as caspases 3 e 8, e ambas participaram do processo de apoptose induzido por dexametasona.

Induction of apoptosis by Korean medicine Gagam-whanglyun-haedoktang through activation of caspase-3 in human leukemia cell line, HL-60 cells.

Gagam-whanglyun-haedoktang (GWH) is a newly designed herbal drug formula based on the traditional oriental pharmacological knowledge for the purpose of treating tumorous diseases. Apoptosis is an evolutionarily conserved suicide program residing in cells. In the present study, apoptosis inducing activities of the decocted water extract of GWH were studied. Results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that GWH had a strong cytotoxic effect on HL-60 cells. The number of live cells was less than 20% after exposure to 1 mg/ml GWH for 48 h. GWH increased cytotoxicity of HL-60 cells in a dose- and time-dependent manner. The percentage of apoptotic cells by flow cytometric analysis of the DNA-stained cells increased to 28%, 31%, and 37% at 24 h and to 37%, 44%, and 81% at 48 h after treatment with 0.01, 0.1, and 1 mg/ml GWH, respectively. DNA fragmentation also occurred in apoptosis and was characterized by a ladder pattern on agarose gel. In addition, GWH increased the secretion of tumor necrosis factor-alpha. GWH-induced apoptosis was accompanied by activation of caspase-3. These results suggest that GWH induces activation of caspase-3 and eventually leads to apoptosis.

HPMA copolymers containing doxorubicin bound by a proteolytically or hydrolytically cleavable bond: comparison of biological properties in vitro.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin bound either by a proteolytically degradable bond (non-targeted PK1 or targeted with alpha-CD71 mAb) or by a hydrolytically degradable bond were synthesised and tested in vivo for various biological properties. Mouse 38C13 B-cell lympoma was used as a well established and defined cell line for this study. 38C13 cells are sensitive to free doxorubicin and IC50 was very low, about 0.014 microM. PK1 showed a strongly decreased cytostatic effect, IC50 being 12.6 microM. alpha-CD71 targeted conjugate, which can be considered as an antibody-targeted form of PK1, had IC50 0.358 microM. HPMA copolymer with doxorubicin bound via a hydrolytically sensitive bond (HYD conjugate) showed a high cytostatic effect with IC50 about 0.052 microM. We demonstrated that HYD conjugate inhibited DNA synthesis and induced p21(Waf1/Cip1) protein expression (p21(Waf1/Cip1) is cyclin-dependent kinase inhibitor which blocks cell cycle progression) as quickly as free doxorubicin, whereas PK1 acted much more slowly. Similarly, apoptosis induction measured by Annexin V binding and Caspase 3 activity was detected later after incubation of cells with PK1 or alpha-CD71 targeted conjugate. Apoptosis was manifested by elevation of bax and bad mRNA levels, which was much more rapid and intense in the case of free doxorubicin and HYD conjugate. Expression of antiapoptotic genes as well as cyclin-dependent kinases was surprisingly not affected.

Mistletoe lectin induces apoptosis and telomerase inhibition in human A253 cancer cells through dephosphorylation of Akt.

Mistletoe lectin has been reported to induce apoptosis in different cancer cell lines in vitro and to show antitumor activity against a variety of tumors in animal models. We previously demonstrated the Korean mistletoe lectin (Viscum album var. coloratum, VCA)-induced apoptosis by down-regulation of Bcl-2 and telomerase activity and by up-regulation of Bax through p53- and p21-independent pathway in hepatoma cells. In the present study, we observed the induction of apoptotic cell death through activation of caspase-3 and the inhibition of telomerase activity through transcriptional down-regulation of hTERT in the VCA-treated A253 cells. We also observed the inhibition of telomerase activity and induction of apoptosis resulted from dephosphorylation of Akt in the survival signaling pathways. In addition, combining VCA with the inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) upstream of Akt, wortmannin and LY294002 showed an additive inhibitory effect of telomerase activity. In contrast, the inhibitor of protein phosphatase 2A (PP2A), okadaic acid inhibited VCA-induced dephosphorylation of Akt and inhibition of telomerase activity. Taken together, VCA induces apoptotic cell death through Akt signaling pathway in correlated with the inhibition of telomerase activity and the activation of caspase-3. From these results, together with our previous studies, we suggest that VCA triggers molecular changes that resulting in the inhibition of cell growth and the induction of apoptotic cell death of cancer cells, which suggest that VCA may be useful as chemotherapeutic agent for cancer cells.

Rotenone-induced apoptosis is mediated by p38 and JNK MAP kinases in human dopaminergic SH-SY5Y cells.

Rotenone is a naturally derived pesticide that has recently been shown to evoke the behavioral and pathological symptoms of Parkinson's disease in animal models. Though rotenone is known to be an inhibitor of the mitochondrial complex I electron transport chain, little is known about downstream pathways leading to its toxicity. We used human dopaminergic SH-SY5Y cells to study mechanisms of rotenone-induced neuronal cell death. Our results suggest that rotenone, at nanomolar concentrations, induces apoptosis in SH-SY5Y cells that is caspase-dependent. Furthermore, rotenone treatment induces phosphorylation of c-Jun, the c-Jun N-terminal protein kinase (JNK), and the p38 mitogen activated protein (MAP) kinase, indicative of activation of the p38 and JNK pathways. Importantly, expression of dominant interfering constructs of the JNK or p38 pathways attenuated rotenone-induced apoptosis. These data suggest that rotenone induces apoptosis in the dopaminergic SH-SY5Y cells that requires activation of the JNK and p38 MAP kinases and caspases. These studies provide insights concerning the molecular mechanisms of rotenone-induced apoptosis in neuronal cells.

Calpain and caspase: can you tell the difference?

Both necrotic and apoptotic neuronal death are observed in various neurological and neurodegenerative disorders. Calpain is activated in various necrotic and apoptotic conditions, while caspase 3 is only activated in neuronal apoptosis. Despite the difference in cleavage-site specificity, an increasing number of cellular proteins are found to be dually susceptible to these cysteine proteases. These include alpha- and beta-fodrin, calmodulin-dependent protein kinases, ADP-ribosyltransferase (ADPRT/PARP) and tau. Intriguingly, calpastatin is susceptible to caspase-mediated fragmentation. Neurotoxic challenges such as hypoxia-hypoglycemia, excitotoxin treatment or metabolic inhibition of cultured neurons result in activation of both proteases. Calpain inhibitors can protect against necrotic neuronal death and, to a lesser extent, apoptotic death. Caspase inhibitors strongly suppress apoptotic neuronal death. Thus, both protease families might contribute to structural derangement and functional loss in neurons under degenerative conditions.