| Objective:The incidence of disease and mortality due to the deteriorating global environment is increasing every year.Human behavior interacts closely with the environment in which we live,and the environment is critical to human health.Studies have shown that the natural environment is a"natural prescription"and that exposure to natural environments(forests,waterfalls,lakes,wetlands,or natural wood materials,etc.)can improve mood,regulate physiological indicators,and prevent disease.In this study,the forest environment of Huangguoshu Waterfall in Anshun City,Guizhou(hereafter referred to as Cascade Forest Environment)was used as the study site,and the park grass and city street environment were set up as the control.A combination of field environmental monitoring and population field experiments and animal experiments was used.The environmental parameters of the Cascade environment were collected,and the physical and mental states of the healthy and subhealthy subjects were observed in the Cascade environment,and the liver inflammation and antioxidant indexes of chronic stress rats were verified by testing.The aim is to investigate the positive effect of the Cascade environment on human health and provide scientific basis for its comprehensive development and utilization.Methods:(1)Investigation and collection of environmental parameters in Cascade Forest:August 2019 was used as the observation period,and the air pollutants(PM2.5),ambient noise values,wind speed,temperature,humidity and negative oxygen ion concentration were collected every two hours from 09:00 to 17:00 daily using portable air quality detectors,negative oxygen ion detectors,humidity and wind speed detectors and other equipment in Cascade Forest environment,park grass environment and urban street environment.),ambient noise values,wind speed,temperature,humidity and negative oxygen ion concentration for 14 consecutive days.When measuring the relevant data within each location,two staff members work together to ensure that the three monitoring sites are tested within the same time frame.All the monitoring data were collected,the wind speed,humidity and temperature data values were substituted into the formula to calculate the temperature and humidity index,wind chill index and dressing index,and the values were assigned against the climate comfort study table,and finally the climate comfort of the three monitoring sites was calculated.The final analysis of the three monitoring sites from the air cleanliness,sound quietness,climate comfort and mixed negative oxygen ion concentration four levels into the situation.(2)The effect of waterfall forest environment on human emotional perceptions and preferences:subjects were recruited online and on site to experience the environment at different test sites.Personal information was filled in to capture the basic information,living habits and general health status of the subjects.A portable eye-tracking device was used to collect subjects’eye-tracking paradigms in the waterfall forest environment,park grass environment and city street environment,to draw gaze hotspot maps and eye-tracking trajectories,and to obtain the Area of Interest(AOI)of environmental elements to screen the landscapes of interest.After each landscape tour,the subjects filled out the Profile of Mood States(POMS)to assess the effect of different environments on the group’s mood,the Perceived Resilience Scale(PRS)to assess psychological changes,and the Likert Environmental Preference Scale to assess preference levels.(3)Effects of waterfall forest environment on psychological,physiological and metabolite levels in subhealthy people:volunteers with chronic fatigue syndrome(CFS,Subhealth)were recruited from the People’s Hospital of Guizhou Province and the Department of Psychosomatic Diseases of Guizhou Medical University,and subjects’basic conditions,lifestyle habits and general health status were collected.A scale was filled out to assess the subjects’psychological and cognitive levels before the start of the experiment.The subjects were randomly divided into a waterfall forest environment group and an urban landscape control group.Each group was exposed to the waterfall forest environment and the urban landscape area for 3 hours per day for a total of 7 consecutive days,respectively.Food and lodging were arranged by the subject researchers during the experiment,and each volunteer wore a uniform brand sports bracelet to monitor daily activity steps and sleep.Individual scores were assessed on the FS-14 Fatigue Scale,Hamilton Depression and Anxiety Scale and PASAT Cognitive Inventory before and after collecting environmental recreation.Peripheral blood was drawn from subjects in both groups at baseline,day 3 and day 7 of the experiment for cortisol(CORT),pentraxin(5-HT),glucose(GLU),triglycerides(TG),total cholesterol(CHO)and uric acid(UA),antioxidant(GSH-Px,SOD,MDA)and inflammatory markers(IL-1β,TNF-α,IL-6,IL-10)etc.were compared.We also used ultra-high pressure liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry(UHPLC-Q-TOF MS)to perform a full-spectrum assay on serum samples from subjects in the Cascade environment to analyze the differential levels of non-targeted metabolites in the serum of subjects at different intervention times.Effect of waterfall forest environment on liver proteins in rats with chronic stress compound fatty liver model:To better fit the chronic stress and poor lifestyle characteristics of chronic fatigue syndrome population in real environment.Forty6-week-old,SPF-rated male SD rats were randomly divided into four groups:normal control group(NC group,n=10),compound model group(CM group,n=10),waterfall forest group(WF group,n=10),and natural recovery group(NR group,n=10).The chronic stress model was established by cold water stimulation and daily swimming in the WF and NR groups,and the success of modeling was evaluated by sugar water consumption and tail suspension test.The rats were fed with high-fat and high-sugar diets to build a fatty liver model,and the degree of liver steatosis was assessed by HE staining and oil red O staining.After successful modeling,the WF group was placed in the Huangguoshu Waterfall Forest environment,and the NR group was placed in the conventional animal center environment and continued to be kept for 7 days.During the experiment,the mental status and weight changes of rats in each group were observed.At the end of the intervention,the behavioral changes of rats were observed using sugar water consumption,hanging tail immobility and exhaustion swimming time.After intraperitoneal anesthesia,blood and tissues and organs were obtained from each group of rats.changes in serum levels of cortisol(CORT),adrenocorticotropic hormone(ACTH),glutathione(GSH),superoxide dismutase(SOD),reactive oxygen species(ROS)and malondialdehyde(MAD)were measured by ELISA.The changes of serum inflammatory factor indicators IL-2,IL-4 and TNF-αwere also detected.The expression levels of glutathione peroxidase(GPx)and superoxide dismutase(SOD)nucleic acids in the hypothalamus,lung,heart,liver,stomach and muscle of different groups of rats were measured by quantitative real-time fluorescence PCR(RT-PCR).RT-PCR was used to determine the levels of reactive oxygen species(ROS)in liver cell suspensions.The relative expression changes of nikamide adenine dinucleotide phosphate oxidase 4(NOX4),Nemo-like kinase(NLK),Toll-like receptor 4(TLR-4),nuclear factorκB inhibitor protein(IκB)and nuclear factorκB(NF-κB)P65 were determined in liver tissues of each group.Immunofluorescence staining laser confocal microscopy was performed to observe the expression of NOX4 and NF-KB P65 displacement in liver sections of each group.western-blot method was used to detect the protein changes of NOX4,TLR4 and downstream NF-κB signaling pathway in liver tissues of each group.Results:(1)Environmental measurements in the field revealed a negative correlation variation between daytime air cleanliness and PM concentration.The air cleanliness was significantly different in the three monitoring sites:the best in the waterfall forest,the second best in the park meadow,and the lowest in the city street.The waterfall forest environment had higher noise values due to the sound of large waterfall drops,animal calls and crowd sound sources.During the continuous testing period,the waterfall forest environment had more days to reach the best comfortable climate than the remaining two monitoring sites.In the waterfall forest environment,there is a mixture of negative oxygen ions produced by waterfall dynamic water molecular cleavage and forest vegetation photosynthesis,and the daytime concentration is relatively stable with no significant trend.Except for a slight decrease in the concentration of negative oxygen ions during the midday hours,the concentration of negative oxygen ions reached above 10,000 ions/cm~3during the rest of the collection time.(2)Participants’eye movement patterns were analyzed by oculomotor:waterfalls were the most interesting environmental element for subjects,and subjects gazed for an increased duration of dwell time and number of return glances compared to other attractions.Blink frequency was also reduced compared to other environments.Compared to the baseline level,the most significant decrease(p<0.01)was observed in the fatigue dimension score on the POMS Mood Scale.The"charismatic"nature of the Cascade environment was well perceived by the subjects.The inner experience was more tranquil,and there were emotional modules of"overlookedness"and"sociability",and the two dimensions were coupled to produce strong attachment emotions.(3)The fatigue scale(FS-14),Hamilton anxiety(HAMA)and Hamilton depression(HMAD)scores of subjects with chronic fatigue syndrome showed a decreasing trend with the prolongation of the waterfall environment recreation time,and the decrease of HAMA was more significant(P<0.01),and the level of neurotransmitter index CORT decreased and the level of 5-HT increased after the waterfall forest environment intervention.Compared with the urban landscape control group,the level of uric acid was reduced in the waterfall forest group.Serum inflammatory(IL-1β,TNF-α)and infection(IL-6,IL-10)related indexes showed a decreasing trend in subjects after recuperation.The metabolomic results suggested that the level of endogenous antioxidants represented by L-ascorbic acid(L-Ascorbic acid)increased significantly(P<0.01)with the increase of recuperation days in the Cascade Forest environment.(4)Compared with the CM group,the rats in the WF group had increased sugar water consumption,decreased immobility time of hanging tail and prolonged exhaustion swimming time;meanwhile,the serum levels of cortisol(CORT)and adrenocorticotropic hormone(ACTH)in the rats in the WF group were significantly decreased(P<0.01).Compared with the CM group,the serum oxidation indexes GSH and SOD levels increased and ROS and MAD levels decreased in the WF group;the expression of inflammation-related indexes IL-1βand TNF-αwas weakened and the expression of anti-inflammatory factor IL-4 increased in the WF group.RT-PCR detection of GPx and SOD nucleic acids in the organ tissues of rats in each group showed that the expression of liver nucleic acids was significantly increased(P<0.01),suggesting that the waterfall forest environment could enhance the content of antioxidant substances in the liver of the compound model rats.Liver single cell suspension reactive oxygen species(ROS)fluorescence staining results showed that ROS fluorescence intensity was significantly reduced in the WF group compared with the NC group.Immunofluorescence of liver sections suggested that the fluorescence expression of NOX4 and NF-κB P65 was significantly enhanced in the CM group compared with the NC group.In the WF group NOX4 fluorescence expression was diminished and NF-κB P65 fluorescence expression in the nucleus was significantly decreased.Meanwhile,RT-PCR results showed that the mRNA expression levels of NOX4,NEMO,TLR4,IκB and NF-κB P65 in the WF group were lower than those in the CM group.In the Western blot experiment,the protein expression of NOX4,NEMO,p-P65,pIκB,TLR4 and IL6 was attenuated in the WF group,and the protein expression level of NLK was increased.Conclusion:(1):Huangguoshu Waterfall forest environment has high climate comfort endowment and good air cleanliness.The air has a stable and high concentration of mixed negative oxygen ions,which is an ideal recreational environment.(2):The charm of the waterfall forest landscape can be perceived by the physical,activities in this environment,there will be a quiet implicit emotional experience,making the individual fatigue significantly reduced,rapid recovery of energy.(3):Huangguoshu waterfall forest environment has been shown to enhance the content of endogenous antioxidant substances in the human body and stabilize the metabolism of choline and amino acids in the body.It has the effect of regulating excessive stress and promoting psychological health for fatigued people.(4):Huangguoshu Waterfall forest has been shown to improve despair and abnormal accumulation of hepatic ROS in compound model rats,increase the level of antioxidant indexes in serum of rats,and decrease the level of pro-inflammatory factors.Long-term housing in this environment may reduce liver damage exacerbated by chronic stress in rats,and inhibition of activation of NOX4/ROS/NF-κB signaling pathway is one of the mechanisms by which the effect is produced. |
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