Altered Expression Profiling of Spinal Genes Modulated by Compound 48/80 in A Mouse Itch Model

Abstract

Background: The molecular mechanisms underlying itch are constantly being studied to create new opportunities to prevent or alleviate itch. The aim of our study was to determine the spinal gene expression changes induced by compound 48/80 in a mouse itch model. Methods: Mice were divided into saline group (control group, n=12) and compound 48/80 group (48/80 group, n=12). 100 µl saline or compound 48/80 was microinjected intradermally in the nape of the neck. After injection, pruritic behavior was immediately measured every 5 minutes. 30 minutes after injection, tissue was prepared to carry out mRNA profiling microarray and reverse transcription polymerase chain reaction (RT-qPCR) analyses. Results: The total numbers of scratching bout after compound 48/80 injection (100 µg/100 µl) were significantly increased in 48/80 group (190 ± 11.33) as compared with control group (6.83 ± 1.17). We screened the dorsal part of the cervical spinal cord of the mouse itch model for differentially expressed genes. Out of 45037 studied transcripts, the abundance levels of 15 transcripts were altered following compound 48/80 injection. 9 and 6 genes were up-and down-regulated in 48/80 group, respectively. We validated the reliability of the microarray results by RT-qPCR, and found 6 up-regulated mRNA, including Sgk1, Bag4, Fos, Ehd2, Edn3 and Wdfy, were significantly increased, whereas 3 down-regulated mRNA, including Corin, 4921511E18Rik and 4930423020Rik, were significantly decreased. Conclusions: These findings indicate that the alterations of spinal gene expression are involved in acute itch, and provide a translational bridge for spinal drugs targeting their signaling pathway to prevent or alleviate itch.

Altered expression of target genes of spinal cord in different itch models compared with capsaicin assessed by RT-qPCR validation

“…The comparative CT method with the formula for relative fold-change = 2 -∆∆CT was used to quantify the amplified transcripts. The specific forward (F) and reverse (R) primer sequences (Table 1 ) were designed [ 1 ]. Experiments were evaluated in triplicate.…”

“…Some patients with serious skin diseases such as atopic dermatitis, advanced liver diseases or renal diseases, will experience moderate to severe itching. Obstinate pruritus is a pathological condition that affects skin sensory processing [ 1 – 4 ]. The mechanisms responsible for intractable pruritus are poorly understood, but refractory itching seems to be enhanced by a state of spinal hypersensitivity.…”

Identification of lncRNA and mRNA expression profiles in rat spinal cords at various time‑points following cardiac ischemia/reperfusion

“…Then the rats were quickly decapitated to limit animal suffering and minimize the effects on the experimental results, and the T1-T4 spinal cord segments were immediately removed and frozen with liquid nitrogen for 1 min, then stored at -80˚C until required. Total RNA from each animal was quantified using a mirVana miRNA Isolation kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and RNA integrity was assessed according to the manufacturer's protocol, which included standard denaturing agarose gel electrophoresis (35)(36)(37). For RNA-seq, the microarray work was performed by CapitalBio Technology Co. Ltd. (Beijing, China), whereby 6 tissue samples (3 model group samples and 3 control group samples) were used for mRNA and lncRNA microarray analysis (38).…”

“…RT-qPCR analysis. The present study extracted total RNA from the upper thoracic spinal cord segments (T1-T4) (40) using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to our previous research (35)(36)(37). The primers for RT-qPCR were designed based on the lncRNA sequences (Table I), and were synthesized and purified at Invitrogen (Thermo Fisher Scientific, Inc.).…”

Quantitative proteomics reveal the alterations in the spinal�cord after myocardial ischemia‑reperfusion injury in rats

“…Rapid advancements in high-throughput technologies and computational frameworks offer an excellent opportunity to quantify spinal nociception using neuronal activation induced by noxious stimuli. The author's previous study showed that transcriptomics and metabolomics enable the examination of spinal biological systems in unprecedented detail (32)(33)(34)(35)(36). More recently, different patterns were revealed in the metabolic and transcriptional levels of the thoracic spinal cord under myocardial ischemia-reperfusion injury (37)(38)(39).…”