Effects of Hyperthermia on TRPV1 and TRPV4 Channels Expression and Oxidative Markers in Mouse Brain

Abstract

Heat stress increases the core body temperature through the pathogenic process. The pathogenic process leads to the release of free radicals, such as superoxide production. Heat stress in the central nervous system (CNS) can cause neuronal damage and symptoms such as delirium, coma, and convulsion. TRPV1 (Transient Receptor Potential Vanilloid1) and TRPV4 genes are members of the TRPV family, including integral membrane proteins that act as calcium-permeable channels. These channels act as thermosensors and have essential roles in the cellular regulation of heat responses. The objective of this study is to examine the effect of general heat stress on the expression of TRPV1 and TRPV4 channels. Furthermore, oxidative markers were measured in the brain of the same heat-stressed mice. Our results show that heat stress leads to a significant upregulation of TRPV1 expression within 21-42 days, while TRPV4 expression decreased significantly in a time-dependent manner. Alterations in the oxidative markers were also observed in the heat-stressed mice.

Keywords: Central nervous system; Heat stress; Oxidative markers; TRPV channels.

Fig. 1

qRT-PCR details for TRPV1, TRPV4 and GAPDH: melt, amplification, and standard curves which endorse PCR results, and used primers efficiency

Fig. 2

Agarose gel: desired band size on agarose gel (TRPV1 114 bp, TRPV4 161 bp, GAPDH 160 bp) endorse fidelity of qRT-PCR

Fig. 3

Relative RNA expression of TRPV1 and TRPV4 genes: a qRT-PCR analyses show no significance expression of TRPV1 compared to control (p value $>0.05).$ b Time-dependent changes in TRPV4 expression levels showing the significant decrease in 21 and 42 days group compared to control group (p value $<0.05).$ ((n = 9) control group), ((n = 8) 7-day group), ((n = 10) 14-day group), ((n = 8) 21-day group), ((n = 9) 42-day group). ***p < 0.001 as compared to control group

Fig. 4

a Western blot: data were obtained from three independent western blot analyses, β-actin was used as a loading control. (n = 4 in each group). b Protein expression levels of TRPV1 and TRPV4 groups: TRPV1 protein levels showed significant upregulation in 21 and 42 heat intervention groups (p value $<0.05).$ Time-dependent changes in TRPV4 protein levels showed a significant decrease of 21 and 42-day groups, which confirms TRPV4 gene expression data in 21 and 42-day groups. (p value $>0.05)$. *p < 0.05 and ***p < 0.001 as compared to control group

Fig. 5

TRPV1 and TRPV4 immunohistochemical stains: Brain sections were prepared and the immunohistochemical technique was used in the brain map-indicated regions including the amygdala, caudate-putamen, cortex, entopeduncular nucleus, hypothalamus, thalamus, and cerebellum. Brown stained regions indicate the expression of TRPV1 (in the left set) and TRPV4 (in the right set). In both TRPV1 and TRPV4 immunostaining, the most stained regions are accompanied by cerebellum and entopeduncular regions respectively and the lowest stained regions are accompanied by hypothalamus in control group. But during heat stress, this ratio is changed so that, TRPV4 staining density in the cerebellum is lower than caudate-putamen, thalamus, and entopeduncular nucleus and also, TRPV1 staining density in the hypothalamus is not lowest in 14 days heat stress group

Fig. 6

Image J analyses of immunohistochemical stain: Evaluating TRPV1 and TRPV4 protein levels displayed that expression means of TRPV4 in the control group is more than TRPV1. In heat-stressed groups TRPV1 is increased in time-dependent manner in cortex, cerebellum, hypothalamus, and entopedunuclear nucleus, whereas TRPV4 shows time-dependent decrease in cortex and cerebellum [(n = 3 in each group]

Fig. 7

Effects of heat stress on stress oxidative markers changes (n = 6 in each group): a SOD levels decreased significantly in the 21-day group compared to control (p = 0.015). b GPx levels increased significantly in the 21-day group compared to control (p = 0.009). c MDA levels showed a significant increase then decrease in 21 and 42 days groups (p = 0.008, 0.036) respectively. d TAC levels showed no significant alteration among the groups. *p < 0.05 and **p < 0.01 as compared to control group

Fig. 8

Schematic diagram for the linkage between TRPV1 and TRPV4 expression levels, ROS generation, and involved pathways