Bone Marrow Mesenchymal Stem Cells (BMSCs) Transplantation Alleviates Acute Pancreatitis Through Inhibiting Inflammation and Promoting Caspase-8 Apoptosis Pathway

Bone marrow mesenchymal stem cells (BMSCs) are capable of multipolar differentiation and repairing injured tissues. Herein, we aimed to investigate the mechanism by how BMSCs modulate the apoptotic pathway in the acute pancreatitis (AP). In this study, primary BMSCs were cultured and administrated into 10 AP mice while 10 healthy mice were taken as a blank group and 10 AP mice as a control group. The mouse pancreatic tissues were assessed by HE staining and evaluated by pancreatitis score and serum amylase detection. Level of inflammatory factors CRP and TNF-α was measured by ELISA and PIPK1, PIPK3, MLKL and Caspase-8 expression was detected by RT-qPCR and Western blot. The pancreatitis score (7.29±1.36) and the serum amylase score of (453.66±103.67) mu/ml of BMSCs group was significantly higher than that of control group, indicating increased tissue repair after BMSCs treatment. BMSCs group exhibited a higher level of CRP (711.01±115.31) and TNF-α (132.81±22.13) in serum compared to control group (p < 0.05). PIPK1, PIPK3, and MLKL expression in BMSCs group decreased (p < 0.05) whereas Caspase-8 was increased (p < 0.05). On the other hand, BMSCs group presented upregulated PIPK1, PIPK3, and MLKL (p < 0.05) and downregulated Caspase-8 (p < 0.05). In conclusion, BMSCs regulate cell apoptosis by upregulating Caspase-8 expression, and downregulating PIPK1, PIPK3 and MLKL level, thereby alleviating the inflammation in AP.

Bacillus anthracis induces NLRP3 inflammasome activation and caspase-8–mediated apoptosis of macrophages to promote lethal anthrax

Lethal toxin (LeTx)-mediated killing of myeloid cells is essential for Bacillus anthracis, the causative agent of anthrax, to establish systemic infection and induce lethal anthrax. The “LeTx-sensitive” NLRP1b inflammasome of BALB/c and 129S macrophages swiftly responds to LeTx intoxication with pyroptosis and secretion of interleukin (IL)-1β. However, human NLRP1 is nonresponsive to LeTx, prompting us to investigate B. anthracis host–pathogen interactions in C57BL/6J (B6) macrophages and mice that also lack a LeTx-sensitive Nlrp1b allele. Unexpectedly, we found that LeTx intoxication and live B. anthracis infection of B6 macrophages elicited robust secretion of IL-1β, which critically relied on the NLRP3 inflammasome. TNF signaling through both TNF receptor 1 (TNF-R1) and TNF-R2 were required for B. anthracis-induced NLRP3 inflammasome activation, which was further controlled by RIPK1 kinase activity and LeTx-mediated proteolytic inactivation of MAP kinase signaling. In addition to activating the NLRP3 inflammasome, LeTx-induced MAPKK inactivation and TNF production sensitized B. anthracis-infected macrophages to robust RIPK1- and caspase-8–dependent apoptosis. In agreement, purified LeTx triggered RIPK1 kinase activity- and caspase-8–dependent apoptosis only in macrophages primed with TNF or following engagement of TRIF-dependent Toll-like receptors. Consistently, genetic and pharmacological inhibition of RIPK1 inhibited NLRP3 inflammasome activation and apoptosis of LeTx-intoxicated and B. anthracis-infected macrophages. Caspase-8/RIPK3-deficient mice were significantly protected from B. anthracis-induced lethality, demonstrating the in vivo pathophysiological relevance of this cytotoxic mechanism. Collectively, these results establish TNF- and RIPK1 kinase activity–dependent NLRP3 inflammasome activation and macrophage apoptosis as key host–pathogen mechanisms in lethal anthrax.

mtROS Induced via TLR-2-SOCE Signaling Plays Proapoptotic and Bactericidal Role in Mycobacterium fortuitum-Infected Head Kidney Macrophages of Clarias gariepinus

The mechanisms underlying Mycobacterium fortuitum-induced mycobacteriosis remain unexplored. Using head kidney macrophages (HKM) from catfish (Clarias gariepinus), we report that Ca2+ surge across mitochondrial-Ca2+ uniporter (MICU), and consequent mitochondrial ROS (mtROS) production, is imperative for mycobactericidal activity. Inhibition of mtROS alleviated HKM apoptosis and enhanced bacterial survival. Based on RNA interference (RNAi) and inhibitor studies, we demonstrate that the Toll-like receptor (TLR)-2–endoplasmic reticulum (ER) stress–store-operated calcium entry (SOCE) axis is instrumental for activating the mt-Ca2+/mtROS cascade in M. fortuitum-infected HKM. Additionally, pharmacological inhibition of mtROS attenuated the expression of CHOP, STIM1, and Orai1, which suggests a positive feedback loop between ER-stress-induced SOCE and mtROS production. Elevated tumor necrosis factor alpha (TNF-α) levels and caspase-8 activity were observed in HKM consequent to M. fortuitum infection, and our results implicate that mtROS is crucial in activating the TNF-mediated caspase-8 activation. Our results for the first time demonstrate mitochondria as an innate immune signaling center regulating mycobacteriosis in fish. We conclude that M. fortuitum-induced persistent SOCE signaling leads to mtROS production, which in turn activates the TNF-α/caspase-8 axis culminating in HKM apoptosis and bacterial clearance.

Differences of Key Proteins between Apoptosis and Necroptosis

Many different types of programmed cell death (PCD) have been identified, including apoptosis and necroptosis. Apoptosis is a type of cell death that is controlled by various genes. It is in charge of eliminating aberrant cells such as cancer cells, replenishing normal cells, and molding the body as it develops. Necroptosis is a type of programmed cell death that combines necrosis and apoptosis. In other words, it takes on a necrotic appearance, although cells die in a controlled manner. Various investigations of these two pathways have revealed that caspase-8, receptor-interacting serine/threonine-protein kinase 1 (RIPK1), and RIPK3 are crucial proteins in charge of the switching between these two pathways, resulting in the activation or inhibition of necroptosis. In this review, we have summarized the key proteins between apoptosis and necroptosis.

Obeticholic Acid Protects Against Cholestatic Liver Injury Induced by Lithocholic Acid via Inhibiting Exogenous Cell Apoptosis

Background: Lithocholic acid (LCA) is one kind of endogenous bile acids which is a typical index in primary biliary cholangitis (PBC). It could cause severe cholestatic liver injury in rodents. Obeticholic acid (OCA) is a major treatment for PBC. However, its effect and mechanism in LCA-induced liver injury was still unclear beside of bile acid regulation. This study aims to evaluate the hepatoprotective effect and mechanism of OCA against LCA-induced cholestatic liver injury. Results: LCA-induced upregulations of ALT, AST, ALP and TBA were reduced and the bile acid profiles in serum, liver and bile were improved significantly by OCA. This bile acid regulating effect of OCA was mainly based on increasing the expression of bile acid efflux transporters bile salt export pump (BSEP), multidrug resistant associated protein 2 (MRP2), MRP3 and multi-drug resistance 3 (MDR3) instead of bile acid synthesis inhibition. Furthermore, it was found that OCA reduced the activation and expression of Caspase 8/3 signaling pathway without the change of p-MLKL and BAX in LCA-induced cholestatic model. And the inhibition of Caspase 8/3 signaling pathway depended on the activation of Farnesoid X receptor (FXR) to inhibit Caspase 8 cleavage to form a active complex.Conclusions: This study found OCA improved LCA-induced cholestatic liver injury via FXR-induced exogenous cell apoptosis, which provided a new evidence for the application of OCA to ameliorate PBC in clinical.

Ultrasound-Targeted Microbubble Destruction-Mediated Inhibition of Livin Expression Accelerates Ovarian Cancer Cell Apoptosis

Objective. Ultrasound-targeted microbubble destruction (UTMD) technique has recently been developed as a nonviral delivery of gene therapy. This study aimed at investigating the survival and apoptosis of ovarian cancer cell line OVCA-433 by inhibiting Livin expression through ultrasound-targeted microbubble destruction. Methods. We synthesized a targeted microbubble agent for UTMD-mediated shRNA against Livin gene in human ovarian cancer OVCA-433 cells. Lipid microbubbles were conjugated with a luteinizing hormone-releasing hormone analog (LHRHa) by an avidin-biotin linkage to target the ovarian cancer OVCA-433 cells expressing LHRH receptors. The microbubbles were mixed with the recombinant plasmid harboring shRNA-Livin. shRNA-Livin was transfected into OVCA-433 cells upon exposure to 1 MHz pulsed ultrasound beam (0.5 W/cm2) for 8 s. Cell survival was measured by the MTT assay, cell apoptosis by flow cytometry using annexin V/PI double staining, and cell ultrastructure by using the transmission electron microscope. The mRNA and protein expression levels of caspase-3 and caspase-8 were detected by RT-qPCR and western blotting. Results. UTMD-mediated delivery of shRNA-Livin remarkably reduced the survival of OVCA-433 cells but promoted the apoptosis compared with shRNA-Livin alone, shRNA-Livin plus nontargeted microbubbles, and shRNA-Livin plus LHRHa-conjugated microbubbles containing shRNA-Livin with or without exposure to ultrasound pulses. It was also found that UTMD-mediated delivery of shRNA-Livin notably declined the mRNA and protein expression levels of caspase-3 and caspase-8 in OVCA-433 cells compared with shRNA-Livin alone, shRNA-Livin plus nontargeted microbubbles, and shRNA-Livin plus LHRHa-conjugated microbubbles containing shRNA-Livin with or without exposure to ultrasound pulses. Conclusion. Our experiment verifies the hypothesis that ultrasound mediation of targeted microbubbles can enhance the transfection efficiency of shRNA-Livin in ovarian cancer cells.