Waterfall Forest Environment Regulates Chronic Stress via the NOX4/ROS/NF-κB Signaling Pathway

Abstract

Background: Forest therapy has been proven to have beneficial effects on people with depression and anxiety. However, it remains unknown whether the waterfall forest environment (WF) affects the physical and psychological health of patients with chronic fatigue and how the WF regulates chronic stress. Methods: Twenty-four patients with chronic fatigue were randomly divided into two groups: the WF group and the urban (U) group. Scores on the Hamilton Anxiety Scale (HAMA), Hamilton Depression Scale (HAMD), and Fatigue Scale-14 (FS-14) were evaluated before and after environmental intervention. Detection of physiological indexes and inflammatory factor levels and immunological analysis were also performed. In addition, the chronic stress rat model was constructed, and the effects of the WF on hopelessness and liver damage of rats were investigated. Results: Patients with chronic fatigue in the WF group showed a significant decrease in FS-14, HAMA, and HAMD scores compared with the U group. The expression levels of glutathione peroxidase and superoxide dismutase were remarkably higher in the WF group than in the U group. However, the expression levels of malondialdehyde and inflammatory factors (IL-1β, TNF-α, IL-6, and IL-10) were remarkably decreased after the intervention of the WF. In addition, animal experiments confirmed that the WF improved hopelessness, liver damage, and excitability of neurons of chronic stress rats. Mechanistically, the WF reduced the liver damage caused by chronic stress in rats by inhibiting the NOX4/ROS/NF-κB signaling pathway. Conclusions: Collectively, the WF had a positive effect on immune enhancement and physical and psychological health in patients with chronic fatigue and might inhibit chronic stress by regulating the NOX4/ROS/NF-κB signaling pathway.

Keywords: NOX4/ROS/NF-κB signaling pathway; chronic fatigue; chronic stress; immunity; waterfall forest environment.

Figure 1

Effects of the waterfall forest environment (WF) on psychological symptoms and physiological indexes. (A) Hamilton Anxiety Scale (HAMA) and Hamilton Depression Scale (HAMD). (B) Fatigue Scale-14 (FS-14). (C) Effects of the WF on the content of serum glucose (GLU), triglycerides (TG), and total cholesterol (CHO). (D) Effects of the WF on the content of uric acid (UA). (E) Effects of the WF on the level of 5-hydroxytryptamine (5-HT). (F) Effects of the WF on the level of cortisol (CORT). U represents the urban group. **p < 0.01, Day 7 WF vs. Day 7 U.

Figure 2

Effects of the waterfall forest environment (WF) on antioxidant indexes and immunity. (A) Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activity. (B) Malondialdehyde (MDA) content. (C) Effects of the WF on the expression of inflammatory factors. (D) Effects of the WF on immune levels. **p < 0.01, Day 7 WF vs. Day 7 U.

Figure 3

The waterfall forest environment (WF) improves hopelessness of chronic stress rats. (A) Effects of WF on body weight of rats in all the groups. (B) Effects of WF on the consumption of sugar water in all the groups. (C) Effects of WF on immobility time of rats in the tail suspension test. (D) Effects of WF on escape latency of rats in the Morris water maze test. *p < 0.05 and **p < 0.01 vs. the normal control (NC) group. ^#p < 0.05 and ^##p < 0.01 vs. the chronic stress model (CM) group.

Figure 4

Effects of the waterfall forest environment (WF) on the expression of reactive oxygen species (ROS). (A) Effects of the WF on the expression of ROS in serum of rats. *p < 0.05 and **p < 0.01 vs. the normal control (NC) group. ^#p < 0.05 and ^##p < 0.01 vs. the chronic stress model (CM) group. (B) Effects of the WF on the expression of ROS in liver tissues of rats. The production of ROS in the liver tissues was detected by using dichlorofluorescein diacetate (DCFH-DA). Scale bars are 50 μm.

Figure 5

The waterfall forest environment (WF) reduced liver damage by inhibiting the level of NOX4. (A) The expression of NOX4 was detected by immunohistochemical staining. Scale bars are 100 ×. (B) Effects of the WF on the mRNA expression of NOX4. The relative mRNA expression of NOX4 in the liver tissues was detected by qRT-PCR. (C) Effects of the WF on the protein expression of NOX4. The relative protein expression of NOX4 in the liver tissues was detected by western blot. GAPDH was used as an internal control. **p < 0.01 vs. the normal control (NC) group. ^#p < 0.05 and ^##p < 0.01 vs. the chronic stress model (CM) group.

Figure 6

Effects of the waterfall forest environment (WF) on the expression of NOX4 and NF-κB p65. The expression of NOX4 and NF-κB p65 was detected by immunofluorescence staining. Scale bars are 50 μm.

Figure 7

The waterfall forest environment (WF) reduced liver damage by inhibiting the NF-κB signaling pathway. (A) Effects of the WF on the mRNA expression of NLK, TLR4, IKB, and NF-κB p65. The relative mRNA expression of NLK, TLR4, IKB, and NF-κB p65 in the liver tissues was detected by qRT-PCR. (B) Effects of the WF on the protein expression of NF-κB p65, IL-6, p-IKB, and IKB. The relative protein expression of ub-NEMO, ub-NLK, TLR4, NF-κB p65, IL-6, p-IKB, and IKB in the liver tissues was detected by western blot. *p < 0.05 and **p < 0.01 vs. the normal control (NC) group. ^#p < 0.05 and ^##p < 0.01 vs. the chronic stress model (CM) group.