Ruxolitinib induces apoptosis and pyroptosis of anaplastic thyroid cancer via the transcriptional inhibition of DRP1-mediated mitochondrial fission.

Anaplastic thyroid carcinoma (ATC) has a 100% disease-specific mortality rate. The JAK1/2-STAT3 pathway presents a promising target for treating hematologic and solid tumors. However, it is unknown whether the JAK1/2-STAT3 pathway is activated in ATC, and the anti-cancer effects and the mechanism of action of its inhibitor, ruxolitinib (Ruxo, a clinical JAK1/2 inhibitor), remain elusive. Our data indicated that the JAK1/2-STAT3 signaling pathway is significantly upregulated in ATC tumor tissues than in normal thyroid and papillary thyroid cancer tissues. Apoptosis and GSDME-pyroptosis were observed in ATC cells following the in vitro and in vivo administration of Ruxo. Mechanistically, Ruxo suppresses the phosphorylation of STAT3, resulting in the repression of DRP1 transactivation and causing mitochondrial fission deficiency. This deficiency is essential for activating caspase 9/3-dependent apoptosis and GSDME-mediated pyroptosis within ATC cells. In conclusion, our findings indicate DRP1 is directly regulated and transactivated by STAT3; this exhibits a novel and crucial aspect of JAK1/2-STAT3 on the regulation of mitochondrial dynamics. In ATC, the transcriptional inhibition of DRP1 by Ruxo hampered mitochondrial division and triggered apoptosis and GSDME-pyroptosis through caspase 9/3-dependent mechanisms. These results provide compelling evidence for the potential therapeutic effectiveness of Ruxo in treating ATC.

Pre-Injection of Small Interfering RNA (siRNA) Promotes c-Jun Gene Silencing and Decreases the Survival Rate of Axotomy-Injured Spinal Motoneurons in Adult Mice.

Brachial plexus injury is a common clinical peripheral nerve trauma. A series of genes in motoneurons were activated in the corresponding segments of the spinal cord after brachial plexus roots axotomy. The spatial and temporal expression of these genes directly affects the speed of motoneuron axon regeneration and precise target organ reinnervation. In a previous study, we observed the overexpression of c-Jun in motoneurons of the spinal cord ventral horn after brachial plexus injury in rats. However, the relevance of c-Jun expression with respect to the fate of axotomy-induced branchial plexus injury in adult mice remains unknown. In the present study, we explored the function of c-Jun in motoneuron recovery after axotomy. We pre-injected small interfering RNA (siRNA) to knockdown c-Jun expression in mice and examined the effects of the overexpression of c-Jun in motoneurons after the axotomy of the brachial plexus in vivo. Axotomy induced c-Jun overexpression in the ventral horn motoneurons of adult mice from 3 to 14 days after injury. In addition, the pre-injection of siRNA transiently inhibited c-Jun expression and decreased the survival rate of axotomy-injured motoneurons. These findings indicate that the axotomy-induced overexpression of c-Jun plays an important role in the survival of ventral horn motoneurons in adult mice. In addition, the pre-injection of c-Jun siRNA through the brachial plexus stem effectively adjusts c-Jun gene expression at the ipsilateral side.

Time-specific microRNA changes during spinal motoneuron degeneration in adult rats following unilateral brachial plexus root avulsion: ipsilateral vs. contralateral changes.

BACKGROUND: Spinal root avulsion induces multiple pathophysiological events consisting of altered levels of specific genes and proteins related to inflammation, apoptosis, and oxidative stress, which collectively result in the death of the affected motoneurons. Recent studies have demonstrated that the gene changes involved in spinal cord injury can be regulated by microRNAs, which are a class of short non-coding RNA molecules that repress target mRNAs post-transcriptionally. With consideration for the time course of the avulsion-induced gene expression patterns within dying motoneurons, we employed microarray analysis to determine whether and how microRNAs are involved in the changes of gene expression induced by pathophysiological events in spinal cord motoneurons. RESULTS: The expression of a total of 3,361 miRNAs in the spinal cord of adult rats was identified. Unilateral root-avulsion resulted in significant alterations in miRNA expression. In the ipsilateral half compared to the contralateral half of the spinal cord, on the 3rd day after the injury, 55 miRNAs were upregulated, and 24 were downregulated, and on the 14th day after the injury, 36 miRNAs were upregulated, and 23 were downregulated. The upregulation of miR-146b-5p and miR-31a-3p and the downregulation of miR-324-3p and miR-484 were observed. Eleven of the miRNAs, including miR-21-5p, demonstrated a sustained increase; however, only miR-466c-3p presented a sustained decrease 3 and 14 days after the injury. More interestingly, 4 of the miRNAs, including miR-18a, were upregulated on the 3rd day but were downregulated on the 14th day after injury.Some of these miRNAs target inflammatory-response genes in the early stage of injury, and others target neurotransmitter transport genes in the intermediate stages of injury. The altered miRNA expression pattern suggests that the MAPK and calcium signaling pathways are consistently involved in the injury response. CONCLUSIONS: This analysis may facilitate the understanding of the time-specific altered expression of a large set of microRNAs in the spinal cord after brachial root avulsion.

Lithium enhances survival and regrowth of spinal motoneurons after ventral root avulsion.

BACKGROUND: During the clinical treatment of the brachial plexus root avulsion (BPRA), reimplantation surgery can not completely repair the motor function of the hand because the axonal growth velocity of the spinal motoneurons (MNs) is too slow to re-innervate the intrinsic hand muscles before muscle atrophy. Here, we investigated whether lithium can enhance the regenerative capacity of the spinal MNs in a rat model of BPRA. RESULTS: The avulsion and immediate reimplantation of the C7 and C8 ventral roots were performed and followed with daily intraperitoneal administration of a therapeutic concentrationof LiCl. After a 20 week long-term rehabilitation, the motor function recovery of the injured forepaw was studied by a grasping test. The survival and regeneration of MNs were checked by choline acetyltransferase (ChAT) immunofluorescence and by Fluoro-Gold (FG) retrograde labeling through the median and ulnar nerves of the ventral horn MNs. The number and diameter of the nerve fibers in the median nerve were assessed by toluidine blue staining. Our results showed that lithium plus reimplantation therapy resulted in a significantly higher grasping strength of the digits of the injured forepaw. Lithium plus reimplantation allowed 45.1% ± 8.11% of ChAT-positive MNs to survive the injury and increased the number and diameter of nerve fibers in the median nerve. The number of FG-labeled regenerative MNs was significantly elevated in all of the reimplantation animals. Our present data proved that lithium can enhance the regenerative capacity of spinal MNs. CONCLUSIONS: These results suggest that immediate administration of lithium could be used to assist reimplantation surgery in repairing BPRA injuries in clinical treatment.

Effects of sevoflurane and propofol on cultured bone-marrow mesenchymal stem cells of rats.

OBJECTIVE: Bone marrow mesenchymal stem cell (BMSC) is a potentially effective vehicle for the cell and gene therapy in clinical disease treatment. We studied whether the most commonly used anesthetic drugs have negative effects on rat BMSCs in vitro. MATERIALS AND METHODS: The cultured BMSCs were treated with sevoflurane (in 1.7%, 2.3%, and 3%); propofol (5 µg/ml, 10 µg/ml and 20 µg/ml); or 2.3% sevoflurane plus 10 µg/ml propofol. After 4-hour treatment, the cultured BMSCs were prepared for MTT reduction assays and cell morphology observation. RESULTS: Compared to the controls, the 4-hour sevoflurane exposure resulted in decreased cell viability of BMSCs in a concentration-dependent manner; however, 1.7% sevoflurane did not reduce the cell viability. The 4-hour propofol treatment did not affect the cell viability; but combined usage of 2.3% sevoflurane and 10 µg/ml propofol decreased cell viability. In BMSCs treated with higher concentration of sevoflurane (1.7% and 2.3%) and combined usage of the two anesthetics, the cell became raritas with wizened cytoplasm and had fewer connections to each other of BMSCs. More than 2.3%, or 2.3% sevoflurane plus 10 µg/ ml propofol caused cytotoxicity to BMSCs. However, propofol up to 20 µg/ml did not harm the BMSCs. CONCLUSIONS: The study indicates that it is necessary to choose the right anesthesia during the BMSCs transplantation therapy.

Neonatal exposure to sevoflurane causes apoptosis and reduces nNOS protein expression in rat hippocampus.

A growing number of studies have shown that commonly used anesthetic agents may cause neurohistopathological changes and persistent behavioral impairments in the developing brain. The effects of sevoflurane, a widely used substance in pediatric anesthesia, on the developing brain have not been thoroughly analyzed thus far. In this study, Sprague-Dawley rats at postnatal day (P)7 were exposed to 2.3% sevoflurane for 6 h continuously. Six hours after exposure, hematoxylin and eosin (H&E) staining was used to determine the morphological changes in the hippocampus. The expression levels of neuronal nitric oxide synthase (nNOS) and caspase-3 in the hippocampus of the pups were determined by immunofluorescence. A single 6-h sevoflurane exposure at P7 caused morphological changes in the hippocampus. Sevoflurane also significantly increased the expression of cleaved caspase-3 and reduced the expression of the nNOS protein. These results indicate that neonatal exposure to sevoflurane causes neurohistopathological changes, apoptosis and decreases nNOS protein levels in the rat hippocampus.

Activation of phospholipase-Cγ and protein kinase C signal pathways helps the survival of spinal motoneurons injured by root avulsion.

The signaling transduction processes involved in avulsion-induced motoneuron (MN) death have not been elucidated. Using the brachial plexus root avulsion rat model, we showed that avulsion-activated phosphorylation of phospholipase-Cγ (PLCγ) and protein kinase C (PKC) occurred in injured spinal MNs within 72 h of injury. Moreover, some MNs positive for PLCγ and PKC are also positive for avulsion-induced neuronal nitric oxide synthase (nNOS). Inhibition of PLCγ/PKC signal pathway, either with PLCγ inhibitor, 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione, or with PLCγ siRNA augmented avulsion-induced MN death. 1-[6-((17ß-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione also inhibited PKC phosphorylation and exacerbated avulsion-induced reductions in the nNOS protein level in injured spinal segments. Moreover, activation of PLCγ/PKC signal pathway with PKC activator, phorbol-12-myristate-13-acetate, decreased avulsion-induced MN death. The temporal profile of PLCγ/PKC signaling appears to be crucial for the survival of spinal MNs after root avulsion. Our data suggest that PLCγ mediates, while PKC and nNOS are associated with, the avulsion-induced MN death in brachial plexus root avulsion.