Protective Effects Of Long-term Hydrotherapy On Chronic Stress And Stress-related Injury In Rats And Mechanisms

BackgroundStress is closely related to human health.While moderate stress benefits to the body by mobilizing the defense mechanism,continuous excessive chronic stress will have adverse effects on physiology,psychology and behavior,and even cause a variety of stress diseases.Sustained high intensity of stress can cause damage to the body,affect individual health,reduce work efficiency,lead to fatigue,and even induce a variety of diseases and extreme events,such as sudden death.Studies show that 75 to 90 percent of huma n diseases are related to stress.Cardiovascular is the primary target organ for stress induced injury.Various animal models of stress(such as noise,electric shock,bondage,dampness and heat,exhaustion swimming,etc.)have found that stress can cause pathological changes in myocardium and vascular endothelial cells,cause myocardial injury and vascular endothelial dysfunction,and cause or aggravate cardiovascular diseases.As a special group,pilots have to deal with a series of aviation special environments(noise,vibration,acceleration,lack of oxygen,etc.)and special circumstances(thunderstorm winds and other special weather,the air of the engine and other special events)for a long time,which cause various psychological physiological stresses.Pilots have to face with more complex stress factors with more powerful stimulus intensity and more serious damage.Although the flight personnel pass through the layer upon layer strict health selects,the cardiovascular disease occurs from time to time.Moreover,the cardiovascular disease consequence is serious,reduces the flight life span,and even endangers the flight safety.The investigation found that the incidence of hypertension in military pilots in China is 2.5-9.8%,and the number of military pilots with pre-hypertension accounts for 38.1%.However,there is a relative lack of research on the mechanism and protective measures of flight stress injury,and it is far from the needs of the rapid development of aviation industry.Especially in China,the intervention means of stress injury mainly include mental health intervention,while medical care is complementary,resulting in the lack of non-drug measures.Taken together,looking for a safe,noninvasive,simple,rapid,effective method with anti-stress and anti-fatigue effect appears to be an urgent and important task.Hydrotherapy,as a traditional rehabilitation therapy,is marked by non-invasive,safe and efficient.It mainly uses the temperature,mechanical and chemical stimulation produced by water on the human body to achieve the purpose of treatment and prevention of diseases.At present,hydrotherapy has developed rapidly in the field of sports medicine and rehabilitation medicine.Clinical practice shows that hydrotherapy has obvious beneficial effects on the prevention and rehabilitation of hypertension,rheumatoid arthritis,cerebral palsy and other diseases,as well as sub-health,and has also been applied to the recovery of fatigue and injury of high-level athletes after exercise.It may be mainly related to the warm effect of water to promote blood circulation,regulate cell metabolism,induce the synthesis and release of heat shock protein,and reduce oxidative stress.However,it is unclear whether hydrotherapy intervention with different temperatures can alleviate chronic stress and cardiovascular damage.Although about the application of hydrotherapy with a long history in China,the role and application of hydrotherapy in modern medicine and military medicine has not been fully understanding for a l ong time.Studies have found a series of changes of human physiological and biochemical indexes after the hydrotherapy at home and abroad,however,the systematic basic theory research is still lacking,and the biological effect and mechanism of hydrotherapy has not been fully elucidated.Therefore,this study is aimed to build a chronic stress-hydrotherapy intervention rat animal model,observe the effects of different temperature hydrotherapy intervention on chronic stress injury,especially cardiovascular injury,explore the optimal anti-chronic stress injury hydrotherapy intervention program,and study its mechanism.Aims1.To observe the protective effect of different temperature hydrotherapy intervention on chronic stress injury in rats.2.To clarify the protective effect and mechanism of specific hydrotherapy intervention on the cardiovascular in chronic stress.Methods1.Chronic stress animal model: 8-week-old adult male Sprague-Dawley rats were used to induce rat stress model with noise compound foot electric shock stimulation.The current intensity was 3 ~ 5 mA,random interval 2 ~ 20 s,continuous electric shock 1 ~2 s,continuous noise(200 ~ 2000 Hz,80 ~ 100 dB)continued to be given during the night of random shock,and 4 hours of electric shock per day(2 hours each time),noise stimulation for 4 hours,a total of 28 day.2.Hydrotherapy intervention program: The stress stimulation of rats is finished every day.After 1 hour of free diet,the hydrotherapy treatment is given.The hydrotherapy is carried out in a special plastic cylinder(20 cm in diameter and 60 cm in height)with a water depth of 10 to 25 cm,10 minutes in water each time.The water temperature is set according to the specific experimental grouping scheme,wherein the hot water immersion(HWI)temperature is 38~40°C;the cold water immersion(CWI)temperature is 13~15 °C;the thermoneutral water immersion(TWI)temperature is 34~36°C;contrast water temperature(CWT)hot temperature 38~42°C,cold temperature 13~15°C,hot water 2min,change cold water for 1 min,alternate cycle 3 times,total 10 min.3.Blood pressure was measured noninvasively with a tail-cuff system every 4 days,rats was measured 2 hours after the end of the electric shock.Each rat was measured 5 times and averaged.4.The levels of Cor?NE?AngII?CK and LDH in serum were measured with commercial ELISA kits.5.Functional assessment of murine mesenteric arterioles.Mesenteric arteriole segments were isolated and mounted in a temperature-controlled myograph.An optimal passive tension(2 mN)was applied for 1 hour before the experiments were started.Mesenteric arteriolar segments were precontracted with 1 ?mol/L PE.A dose-response curve was obtained by cumulative addition of Ach/SNP/5-HT.Relaxation at each concentration was measured.6.Behavioral experiment:The open field test(OFT)and the elevated plus maze(EPM)experiment was carried out in a quiet environment.The rats were placed in the center of the bottom of the open field reaction box or the center of the elevated cross maze,and the camera was turned on and timed.The OFT experiment was stopped after 5 min of observation.The EPM experiment was stopped after 5 min.7.The statistical analyses were performed using the GraphPad Prism software,version 8.0.Values are presented as mean ± SD.Values of P < 0.05 were considered statistically significant.Results1.The model of chronic stress injury in rats was successfully constructed by electric foot-shock and noise interventions of 28 days.Compared with the control group,the rats in the stress group showed obvious stress response and myocardial injury,which were mainly manifested as significantl y increased blood pressure and blood glucose(P < 0.05),significantly decreased body weight(P < 0.01),increased serum concentrations of Cor,NE,AngII,TNF-?,ADMA,CK and LDH(P < 0.05),and decreased serum NO(P < 0.05),positive staining of autophagy related protein LC3 B and increased expression levels of LC3II/I,p62 and Beclin1 in myocardial tissue(P < 0.05).The number of myocardial mitochondria decreased and showed structural disorder in the stress group.In addition,the open field test(OFT)and the elevated plus maze(EPM)experimental results demonstrated that loco-motor activity and exploratory behavior in rats significantly decreased and anxiety increased in the stress group.2.The effects of different temperature hydrotherapy intervention on chronic stress in rats were different,and the effect of long-term Hot water immersion intervention on chronic stress was the best.Compared with the stress group rats,we found that just 10 minutes a day in the Hot water immersion intervention can significantly reduce the chronic stress reaction of rat body,including: reduced the weight of stress rats,accelerated the recovery of blood glucose levels after stress,reversed stress rats blood pressure,reduced the anxiety level of stress rats,improved stress rats spontaneous activities and exploratory behavior.In addition,alternating hot and cold water bath can also reverse the increase of blood pressure in stress rats,but the effect is late,the blood pressure decreased until the end of the intervention(24 days).Other water bath temperature had no significant effect on weight,blood pressure,blood glucose and behavior of stressed rats.3.Hot water immersion can effectively improve endothelial function in chronic stress rats.Compared with the stress group,endothelium-dependent vasodilation was significantly improved in the hot-water bath group,serum NO level was increased(P < 0.05),ADMA concentration was decreased(P < 0.05),and eNOS phosphorylation level and DDAH1 expression level were increased in the vascular tissue(P < 0.05).In cultured human umbilical vein endothelial cells(HUVECs)10 minutes each day in 40 ? incubation can better simulate the Hot water immersion on the effect of vascular endothelial,including NO secretion increased in warm incubation stress hormone treated HUVECs,ADMA levels dropped,DDAH1 expression increased,and using DDAH1 siRNA can block DDAH1 expression,thus blocking the NO secretion and increasing ADMA concentration.4.Hot water immersion can effectively reduce myocardial injury in chronic stress rats.Compared with the stress group,the levels of NE,AngII,TNF-?,CK,LDH,p62 and Beclin1 were decreased in the Hot water immersion group(P < 0.05),and the mitochondrial damage was also reversed.Conclusions1.In this study,it was found for the first time that Hot water immersion can effectively alleviate chronic stress state compared with other temperature water bath.2.Hot water immersion reduces the increase of blood pressure induced by chronic stress,and the mechanism is related to the increase of endothelial DDAH1 expression,the decrease of endogenous eNOS inhibitor ADMA generation,and the increase of endothelial NO bioavailability in Hot water immersion.3.Hot water immersion can alleviate myocardial injury caused by chronic stress,and its mechanism may be related to the regulation of autophagy disorder of myocardial cells caused by stress and the promotion of mitochondrial structure recovery.Hot water immersion is expected to be an effective protective means to relieve chronic stress,prevent stress hypertension and myocardial injury.