Heat Acclimation Regulates Autophagy-lysosome Pathway To Protect Brain Injury Caused By Heat Stroke

Background:In recent years, the global heat wave frequency and intensity increased year by year, and led thousands of people suffered from heat stress illness(HSI) every year. Heat stroke(HS) is one of the most serious HSI, its major clinical manifestations included high core temperature(Tc), above 40 ℃, and in combination with the central nervous system(CNS) disorders and multiple organ dysfunction syndrome(MODS). The reported mortality rate of HS was 10%~50%. The soldiers are in high-risk of HS and there are deaths reports every year. Especially when soldiers perform tasks in hotspots, the incidence of HS will increase sharply and the mortality can be as high as 30%-50% if the patients were not treated effectively. So the prevention of thermal damage induced by HS is of great importance to improve the fighting capacity for the forces in the hotspots and it’s also one of the important contents of the conventional health care.Heat acclimation(HA) is one of the important measures to prevent thermal damage to the forces in hotspots. “The health regulations of heat resistance training for soldiers” was issued by the Chinese People’s Liberation Army and ruled the troops in hotspots must perform heat resistance training before performing tasks. HA refers to the adaptive responses induced by long-term and repeated exposed to heat stress, including improved reflective adjusting function to heat stress and better physiological function, such as increased sweating rate, lowered heart rate, Tc and metabolic rate, leading to heat dissipation increasing and heat production declining. The CNS damage is one of the main clinical features in HS, and the main pathology factor of neurological sequelaes caused by HS. Some studies demonstrated that HA could down-regulate the irritability of the nervous system to damage thermal stimulation. But so far, quite a few researchers reported the protection of HA on CNS damage caused by HS. Therefore, it is necessary and of great significance to verify the protection of HA on brain injury incurred in HS and investigates its related mechanism.Autophagy is a kind of highly conservative cell biology behavior, refers to self-eating phenomenon appeared in cells. When the detrimental factors occurred, such as extracellular energy deficiencies, ischemia, hypoxia, high fever and intracellular protein folding errors, protein aggregations, metabolic disorders, organelles aging or burst, the autophagy will be induced. Autophagy is mainly involved in digestion and reuse of macromolecular substances and damaged organelles in the cells, and plays an important role in promoting cell survival, maintaining cellular homeostasis and cell health. In neurons, autophagy protects the nervous system from injury by removing the neurotoxic protein aggregates. However, in certain settings where there is uncontrolled upregulation of autophagy, autophagy can lead to cell death, possibly through activating apoptosis or possibly as a result of the inability of cells to survive the non-specific degradation of large amounts of cytoplasmic contents, referred to as autophagy programmed cell death. Both HA and HS are stimulated by outside environment of elevated temperature, the differences were the forming conditions of HA is long term and repeated mild thermal stimulation, while the HS is fatal high intensity thermal stimulation in a short time. And the physiological effects of HA and HS are completely different, thus we speculated that the autophagy induced by HA and HS and its mechanism of pathological and physiological regulation may be not the same.During the process of autophagy, autophagosome packages the contents which need to be degraded and fusions with lysosomes to form autolysosome, then the packaged content were digested by the cathepsins and hydrolytic enzymes in the lysosome and the products were reused by the cell. Thus, the function of lysosome plays a key role in the process of autophagy, while cells were impaired when sufferring from stress, the lysosome function would be involved in. So far, whether the function of lysosome would be different when suffering the two different intensity stress, heat acclimation and heat stroke, and how it would regulate the autophagy is still unclear.Consequently, this paper was intended to investigate the protection role of HA on brain damage caused by HS and its relationship with autophagy. So as to reveal the mechanism of HA-induced neuroprotection and provide additional references for the prevention and treatment of HS.Methods:1.Healthy male C57BL/6 mice aged 8 weeks were employed to establish the HA model by continuous exposing to(34.5±1.0)℃ and relative humidity(70±5)% for 30 days, and HS model by exposing to(43±0.5)℃ and relative humidity(70±5)% for 120 minutes. Then the mice were divided into healthy control(CON) group, HA group, HS group and HA+HS group, each n=12.2.The protection of HA on brain injury caused by HS was investigated by monitoring and comparing the general behaviors, anal temperature changes, the brain water content and brain tissue pathological changes and nerve cell degeneration and apoptosis among the different groups.3.The effect of autophagy in the protection of HA on brain damage caused by HS was investigated by detecting the expressions of autophagy related proteins LC3 and Beclin-1 and the formation of autophagosome, and by monitoring the effect of autophagy inhibitor 3-methyladenine(3MA).4.The effect of lysosome in autophagy regulation was investigated by detecting the expressions of lysosome associated membrane protein 1(LAMP-1) and Cathepsin B, and the activity of Cathepsin B.Results:1.The HA and HS mice was established successfully by continuous exposing to(34.5±1.0)℃ and relative humidity(70±5)% for 30 days, and by exposing to(43±0.5)℃ and relative humidity(70±5)% for 120 minutes, respectively. The model provided an essential experimental basis for further research.2.HA provided protection on brain damage caused by HS. The evidences included: when exposure in 43℃, the mice received HA-pretreatment appeared a better stare of general behavior, the brain injury symptoms delayed, the highest anus temperature, mortality, brain water content, nerve cell pathological damage degree, nerve cell apoptosis and degeneration degree were all lower than the un-HA mice, and all the differences were statistically significant(all P< 0.05). While there was no difference between the CON and HA group. The results suggest that HA itself is not harmful to the nervous system and HA plays protection on brain damage caused by HS.3.Compared with the CON group, the expressions of autophagy related proteins LC3 and Beclin-1 and the formation of autophagosome in the brain tissue of HA, HS and HA + HS group were all increased significantly(P < 0.05), suggesting that heat stimulation elevated the autophagy level in central nervous cells. Compared with HS group, the expressions of LC3 and Beclin-1 and the formation of autophagosome declined dramatically(P< 0.05), suggesting that HA- pretreatment down-regulated the autophagy level induced by HS. After the application of autophagy inhibitor 3 MA, the autophagy were suppressed in 3MA group and HA+3MA group, while the brain water content and degree of neurons degeneration and apoptosis had no obvious changes, suggesting that 3MA is not harmful to the brain. Compared with HA+HS12h group, the autophagy was also suppressed in HA+3MA+HS12h group, and the brain water content and degree of neurons degeneration and apoptosis were increased significantly(P< 0.05), suggesting that the inhibition of autophagy compromised the HA-induced neuroprotection on HS-caused brain damage. Compared with HS12 h group, both the autophagy and the brain water content and degree of neurons degeneration and apoptosis were not changed in 3MA+HS12h group, suggesting that 3MA did not play a role in HS mice.4.Compared with the CON group, the expressions of LAMP-1 and Cathepsin B, and the activity of Cathepsin B in HA group were elevated slightly, while declined sharply in HS group(P < 0.05), suggesting that the fatal thermal stimulation of HS could destroy the structure and enzyme activity of the lysosome, thus affect the lysosome function. Compared with the HS group, the expressions of LAMP-1 and Cathepsin B, and the activity of Cathepsin B in HA+HS group were higher significant(P < 0.05), especially in the early recovery, the differences were more apparent, suggesting that HA-pretreatment enhanced the function of lysosome, protected against the structure and function injury caused HS.Conclusions:1.Heat acclimatization plays a protective role on brain injury caused by heat stroke;2.Heat acclimation plays neuroprotecions by regulating the heat stroke inducedautophagy level;3.The autophagy-lysosome pathway plays an important role in the protection of heat acclimation on brain injury caused by heat stroke.The fatal thermal stimulation of heat stroke destroyed the normal function of the lysosome, while Heat acclimation increased the lysosome function and protected lysosome from heat stroke injury.