Anti-fatigue Activity Of Gardenia Yellow Pigment And Cistanche Phenylethanol Glycosides Mixture in Hypoxia

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Maoxing Li, et al

Abstract

The plateau, which has a unique geographical environment, forms extremely harsh climate conditions. Compared with that of people in the plains, the athletic ability of people on the plateau is significantly decreased. People who enter the plateau from the plains for short time periods will suffer from metabolic dysfunction, productivity loss, bradykinesia, and a significant decrease in overall exercise endurance, which results in exercise fatigue. To investigate the anti-fatigue activity of gardenia yellow pigment and Cistanche phenylethanoid glycosides mixture under hypoxia, we performed anti-hypoxia, normoxia anti-fatigue, and hypoxia anti-fatigue experiments. The gardenia yellow pigment and Cistanche phenylethanoid glycosides mixture have good anti-hypoxia and anti-fatigue effects under hypoxia, which can significantly prolong the anti-anoxia time of mice, and prolong the exhausted swimming time of mice under normoxic and hypoxic conditions. The mechanisms may be related to reducing the accumulation of bad metabolites, increasing energy reserves, improving free radical scavenging capacity, increasing related metabolic enzymes activities, reducing apoptosis, and inhibiting the infiltration of inflammatory cells. The gardenia yellow pigment and Cistanche phenylethanoid glycosides mixture is a potential functional product to improve hypoxic exercise fatigue.

Keywords:

Gardenia yellow pigment, Cistanche phenylethanoid glycosides, Anti-hypoxia, Anti-fatigue

Cistanche phenylethanoid glycosides have an Anti-fatigue function

1. Introduction

Qinghai-Tibet plateau and Pamirs are known as the “roof of the world”, with an average elevation of over 4000 m. It accounts for more than 25% of China’s total territory. The plateau, which has a unique geographical environment, forms extremely harsh climate conditions. Compared to that of people in the plains, the athletic ability of people on the plateau is significantly decreased. People who enter the plateau from the plains for a short time will suffer from metabolic dysfunction, productivity loss, bradykinesia, and a significant decrease in overall exercise endurance, which results in exercise fatigue. And long-term exposure to the anoxic environment can lead to a series of pathophysiological changes in the nervous system, respiratory system, and circulatory system (Du et al., 2016; Finsterer, 2016; Lee, Kim, Han, Kim, & Son, 2015; Ma et al., 2011). Sports fatigue refers to the body’s ability decline after a long period of strenuous exercise. Fatigue is related to the accumulation of metabolic products, the consumption of energy and substances, the disorder of free radicals, and the imbalance of the internal environment (Y. Chen et al., 2016).

Cistanche deserticola, the dry scaly fleshy stem leaf of Cistanche deserticola Y.C. Ma, can relax the bowels and resist senility (Cao, Zhao, & Wu, 2004). It is a precious medicinal herb that is mainly grown in dry desert areas. Phenylethanoid glycosides are one of the main chemical constituents of Cistanche deserticola and have a wide range of anti-inflammatory, antibacterial, antiviral, antitumor, antioxidant, enhanced memory, immune regulation, and impotence curing effects (Wei & Yingni, 2013; Xue, Yan, & Yang, 2016; Zhou et al., 2016). Gardenia, dry and mature fruit of Gardenia jasminoides Ellis, has many pharmacological activities, including gall bladder and liver protection, anti-inflammatory, and analgesic activities, antibiosis, antitumor activity, and blood sugar and blood lipid reduction activities (J.-F. Chen et al., 2012; Kang, Jin, Oh, & Kim, 2017; Xiangle et al., 2011). Crocin, a potent antioxidant mainly obtained from saffron, also shows high content in gardenia yellow pigment (Liu, Chen, Li, & Zhang, 2012; Soeda et al., 2007). Gardenia yellow pigment has excellent dyeing ability for protein, starch, etc., and can be widely used in various foods, such as cakes, candy, flour, beverages, jellies, biscuits, and ice cream.

Our study mainly investigates the effect of improving the function of a mixture of two components on simulated high-altitude exercise fatigue in rats under hypoxia. Through the study of a mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides, we assessed the feasibility of producing food products with anti-hypoxia and anti-fatigue effects.

2. Methods and materials

2.1. Animal

All BALB/c mice (20 ± 2 g) and Wister rats (200 ± 20 g) used in this experiment were SPF animals and were provided by the Center for Experimental Animals. The mice and rats were kept at 22 ± 2 ◦C. The experiment was approved by the Laboratory Animal Management Committee of the 940th Hospital of Joint Logistic Support Force of PLA (Lanzhou, China).

2.2. Drugs, chemicals, and reagents

The gardenia yellow pigment was purchased from Lubao Biological Technology (Qianjiang, China). The Cistanche phenylethanoid glycosides preparation was purchased from Tairen Biological Technology (Changchun, China). The BCA protein assay kit, Coomassie brilliant blue protein assay kit, Blood Urea Nitrogen (BUN) assay kit, Creatine Kinase (CRE) assay kit, Uric Acid (UA) assay kit, Pyruvate (PA) assay kit, Pyruvate Kinase (PK) assay kit, Lactic Acid (LD) assay kit, Lactate Dehydrogenase (LDH) assay kit, Reduced Glutathione (GSH) assay kit, Glutathione Peroxidase (GSH-PX) assay kit, Total Superoxide Dismutase (T-SOD) assay kit, Catalase (CAT) assay kit, Nitric Oxide (NO) assay kit, Nitric Oxide Synthase (NOS) assay kit, Malonaldehyde (MDA) assay kit, ATP assay kit, and liver/muscle glycogen assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). PMSF and RIPA were purchased from Beyotime Biotechnology Research Institute. The 10 × PBS, 4% paraformaldehyde, 4 × protein sample buffer, Glycine, SDS, TRIS, SDS-PAGE gel preparation kit, 10 × TBST, non-fat powdered milk, and ECL Plus hypersensitivity reagent were purchased from Beijing Solarbio Technology (Beijing, China). A prestained protein ladder was purchased from Thermo Fisher Scientific (MA, USA). Mouse Anti-β-actin, horseradish peroxidase-labeled Goat anti-mouse, and horseradish peroxidase-labeled goat anti-rabbit antibodies were purchased from Golden Bridge Biotechnology. The caspase-3 polyclonal antibody was purchased from Cell Signaling Technology. Anti-Bax antibody, Anti-Bcl-2 antibody, Anti-AMPK alpha1+AMPK alpha2 antibody, and Anti-Nox2/gp91phox antibody were purchased from Abcam (United Kingdom). Immobilon®-P PVDF Transfer Membranes were purchased from MilliporeSigma (MO, USA). Anhydrous methanol and ethanol were purchased from Edward Chemical (Lanzhou, China).

2.3. Closed atmospheric test

Fifty BALB/c mice were randomly divided into the control group (sterile water, 0.1 mL/10 g), positive control group (Rhodiola, 0.5 g⋅kg− 1 ⋅d− 1 ), low-dose group (0.1 g⋅kg− 1 ⋅d− 1 ), middle-dose group (0.3 g⋅kg− 1 ⋅d− 1 ) and high-dose group (0.5 g⋅kg− 1 ⋅d− 1 ). The drugs were given 3 days consecutively. One hour after the last administration, the mice were placed in a 200 mL jar with 5 g of soda lime (absorption of carbon dioxide and water), covering the bottle tightly with Vaseline, and time was counted immediately. The mice were observed and the time when the mice died due to lack of oxygen was recorded.

cistanche bodybuilding

2.4. Sodium nitrite hypoxia test

The animals were grouped and administered the same methods as described in 2.3. One hour after the last administration, 300 mg/kg sodium nitrite solution was injected into the abdominal cavity. The mice were observed and the time when the mice died due to lack of oxygen was recorded.

2.5. Acute hypoxia test

The animals were grouped and administered the same methods as described in 2.3. One hour after the last administration, the mice were placed in the hypobaric hypoxia animal test chamber (altitude: 10,000 m), and the rising speed was 20 m/s. Timing began when the altitude reached 10,000 m. The mortality of the mice was observed and recorded within 15 min.

2.6. Study on the anti-fatigue activity of an equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides in normoxia

Sixty BALB/c mice were randomly divided into a control group (sterile water, 0.1 mL/10 g), a positive control group (Rhodiola, 0.5 g⋅kg− 1 ⋅d− 1 ), and drug groups (low dose, 0.1 g⋅kg− 1 ⋅d− 1 , middle dose, 0.3 g⋅kg− 1 ⋅d− 1 , and high dose, 0.5 g⋅kg− 1 ⋅d− 1 ). The drugs were administered for 7 days continuously. The exhaustive swimming test was carried out, and the time of exhaustive swimming was measured among the mice 1 h after the last administration. The wire (the weight was 7% of the bodyweight of mice) was fastened to 1/3 of the mouse tail, and the mice were placed in 18 cm deep water (50 cm × 40 cm × 30 cm) under the control of water at a temperature of 25 ± 1 ◦C, and the exhaustive swimming test was conducted. The time from putting mice into the water to immersion in water was recorded (when the mouse could not raise itself to the surface for 7 s). After the exhaustive swimming test, whole blood was collected from the mouse orbital, and liver, brain, and muscle tissues were obtained. The levels of PA, BUN, LD, LDH, GSH, T-SOD, liver glycogen, and muscle glycogen were determined. The protein expression of Bax, Bcl-2, and Caspase-3 in liver and brain tissues was measured by western blotting.

2.7. Study on the anti-fatigue activity of an equal proportion mixture of cistanche phenylethanoid glycosides and gardenia yellow pigment in hypoxia

In total, 75 Wistar rats were randomized into 5 groups: normoxic control group (NC, distilled water); hypoxia control group (HC, distilled water); exhaustive swimming control group (EC, distilled water); exhaustive swimming positive control group (EP, 0.5 g⋅kg− 1 ⋅d− 1 ); and exhaustive swimming drug group (ED, 0.5 g⋅kg− 1 ⋅d− 1 ). The rats in the NC group were placed in the animal room at local elevation (Lanzhou, 1500 m), and the remaining rats were placed in a plateau environment at a simulated 8000 m altitude that was administered continuously for 5 days. The specific experimental procedure was the same as that described in 2.6

All rats were decapitated, and brain tissue, liver tissue, and muscle tissue were collected. The levels of PA, BUN, UA, CRE, CK, CAT, GSH-PX, MDA, NOS, NO, LD, LDH, T-SOD, ATP, liver glycogen, and muscle glycogen were measured. Protein expression levels of Bax, Bcl-2, Nox2, and Ampk in liver and brain tissues were measured by western blotting. Two rats in each group were stained with hematoxylin and eosin (HE), and pathological sections were observed.

2.8. Statistical analysis

All data were expressed as the mean ± SD. Data were subjected to analysis of variance (ANOVA) followed by Student–Newman–Keuls tests. P < 0.05 was considered significant.

3. Results

3.1. The anti-hypoxia activity of the equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides

3.1.1. The result of closed atmospheric test

Compared with the control group, the anti-hypoxia time of the positive control group, the low-dose group, middle-dose group and highdose group were prolonged 8.88% (P < 0.05), 7.86% (P < 0.05), 20.74% (P < 0.05) and 22.18% (P < 0.01), respectively. The high-dose group had a more significant anti-hypoxia effect (P < 0.01) (Table 1).

3.1.2. The result of the sodium nitrite hypoxia test

Compared with the control group, the anti-hypoxia time of the positive control group, the low-dose group, middle-dose group and highdose group were prolonged by 16.49% (P < 0.05), 20.83% (P < 0.01), 23.99% (P < 0.01) and 20.28% (P < 0.05), respectively. The low-dose and high-dose drug groups had obvious anti-hypoxic effects (P < 0.01) (Table 2).

3.1.3. The results of acute hypobaric hypoxia

Compared with the control group, within 15 min of when the mice were placed in a hypobaric hypoxia test chamber, the mortality of the positive group and the drug groups decreased obviously, and the rates of decline in the positive control group, low-dose group, middle-dose group, and the high-dose group were 30%, 20%, 30%, and 40%, respectively. The mortality of the mice in the high-dose group was the lowest. The results are shown in Table 3.

3.2. The anti-fatigue activity of an equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides

3.2.1. The exhaustive swimming time

Compared with the control group, the time of exhaustive swimming for the positive control group, low-dose group, middle-dose group, and high-dose group was prolonged by 21.18% (P < 0.05), 11%, 24.35% (P < 0.05) and 26.9% (P < 0.05), respectively (Table 4). The prolongation time was longest in the high-dose group.

3.2.2. Determination of biochemical indexes

The BUN level in the serum of the drug groups was significantly lowered, and the level of PA in serum was increased; the difference was obviously significant compared with that of the control group (P < 0.05) (Fig. 1A). The BUN level in the positive control group, low-dose group, middle-dose group and high-dose group were 26.2% (P < 0.01), 28.2%, 25.4% (P < 0.01) and 25.9% (P < 0.01) lower than those in the control group, respectively. The PA level in the positive control group, low-dose group, middle-dose group and high-dose group was increased by 12.2% (P < 0.05), 9.1%, 9.8% (P < 0.01) and 16.4% (P < 0.01), respectively, compared with that of the control group.

Compared with those of the control group, the levels of LD and LDH in the drug groups increased significantly (P < 0.05) (Fig. 1B; Fig. 1C), and the contents of GSH and T-SOD in the drug groups increased significantly (P < 0.05) (Fig. 1D; Fig. 1E).

Compared with that of the control group, the LD level in the positive control group, low-dose group, middle-dose group and high-dose group in serum was elevated significantly by 17.9% (P < 0.01), 6.3%, 16.5% (P < 0.05) and 18.0% (P < 0.05), respectively. The liver LD level in the positive control group and drug groups was elevated significantly by 58.6% (P < 0.01), 64.9% (P < 0.01) 107.5% (P < 0.01) and 73.6% (P < 0.01), respectively. The brain LD level in the positive control group and drug groups was elevated significantly by 33% (P < 0.01), 22.4%, 31% (P < 0.01) and 3.9%, respectively. The muscle LD level in the positive control group and drug groups was elevated significantly by 31.7% (P < 0.01), 27.5% (P < 0.01), 52.7% (P < 0.01) and 47.6% (P < 0.01), respectively.

Compared with that of the control group, the LDH level in the positive control group, low-dose group, middle-dose group and high-dose group in serum was elevated significantly by 6.1% (P < 0.05), 11.4% (P < 0.05), 19.1% and 1.9% (P < 0.01), respectively. The liver LDH level was elevated significantly by 22.4% (P < 0.01), 22.3% (P < 0.01), 30% (P < 0.01) and 21.7% (P < 0.01), respectively. The brain LDH level was elevated significantly by 21.3% (P < 0.05), 10.5%, 27.5% (P < 0.01) and 31.2% (P < 0.01), respectively. The muscle LDH level was elevated significantly by 5.1%, 4.5%, 11.5% (P < 0.05) and 16.8% (P < 0.01), respectively.

Compared with that of the control group, the liver SOD level in the positive control group, low-dose group, middle-dose group and highdose group was elevated significantly by 8.9% (P < 0.05), 20.6% (P < 0.01), 36.7% (P < 0.01) and 19.3% (P < 0.01), respectively. The brain SOD level in the positive control group and drug groups was elevated significantly by 12.6% (P < 0.01), 8.3%, 7.7% (P < 0.05) and 11% (P < 0.05), respectively. The muscle SOD level in the positive control group and drug groups was elevated significantly by 10%, 46.3%(P < 0.05), 32.6% (P < 0.01) and 19% (P < 0.05), respectively.

Compared with that of the control group, the liver GSH level in the positive control group, low-dose group, middle-dose group and highdose group was elevated significantly by 29.4%, 31.2% (P < 0.01), 47.3% (P < 0.01) and 14.6% (P < 0.01), respectively. The brain GSH level in the positive control group and drug groups was elevated significantly by 40.6% (P < 0.05), 24.6%, 57.1% (P < 0.01) and 57.9% (P < 0.01), respectively. The muscle GSH level in the positive control group and drug groups was elevated significantly by 174% (P < 0.01), 45.4% (P < 0.05), 10.1% and 38.2% (P < 0.01), respectively.

Cistanche phenylethanoid glycosides have an Anti-fatigue function

3.2.3. Detection of the expression of related apoptotic proteins in the liver and brain tissues of mice by western blotting

When compared with those of the control group, Bax/Bcl-2 and Caspase-3 levels in liver tissue of the drug groups and positive control group were obviously reduced (P < 0.05), while levels in the high-dose drug group were obviously decreased (P < 0.01) (Fig. 2a). Bax/Bcl-2 and Caspase-3 levels in brain tissue of the drug groups and positive group were reduced (P < 0.01) (Fig. 2b).

Compared with those in the control group, the Bax/Bcl-2 level in the low-dose group, middle-dose group, high-dose group and positive control group in liver was decreased significantly by 9.8%(P < 0.01), 14.6%, 26.4% (P < 0.01) and 26.6% (P < 0.01), respectively. Compared with that of the control group, the Caspase-3 level in the low dose group, middle dose group, high-dose group and positive control group in liver was decreased significantly by 2.8%, 65.7% (P < 0.01), 76.2% (P < 0.01) and 97.8% (P < 0.01), respectively.

Compared with those in the control group, the Bax/Bcl-2 level in the low-dose group, middle-dose group, high-dose group and positive control group in the brain was decreased significantly by 14.2% (P < 0.05), 38.6%, 24.3% (P < 0.01) and 8.5% (P < 0.05), respectively. Compared with that in the control group, the Caspase-3 level in the low-dose group, middle-dose group, high-dose group and positive control group in the brain was decreased significantly by 32.4% (P < 0.05), 34.2% (P <0.01), 17.5% (P < 0.05) and 8.3%, respectively.

3.3. The improvement effect of the equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides for exercise fatigue at high altitude

3.3.1. The time of exhaustive swimming

The results showed that compared with the EC group, the EP and ED groups were capable of significantly increasing the exhaustive swimming time of mice (P < 0.05) (Table 5). Compared with the EC group, the time of exhaustive swimming in both the EP and ED groups was prolonged by 49.77% and 51.71%, respectively.

3.3.2. The biochemical parameters in serum

Compared with those in the NC group, the serum levels of BUN, CRE, UA, and PA in the other groups were significantly increased (P < 0.01), and those in the HC group were prolonged by 63.1%, 60.7%, 55.8%, and 100%, respectively. Compared with those in the EC group, the levels of BUN, CRE, UA, and PA in serum of EP and ED groups decreased significantly (P < 0.05) (Fig. 3A). Specifically, the levels of BUN, CRE, UA and PA in the EP group were lowered by 8.4%, 23.9% (P < 0.05), 14.3% (P < 0.05) and 10.1%. Further, the levels of BUN, CRE, UA, and PA in the ED group were lowered by 11.7% (P < 0.05), 24.9% (P < 0.05), 10.8% (P < 0.05) and 8.7%. Compared with those in the NC group, the levels of LD, LDH, NO, and NOS in the serum of the other groups were significantly increased (P < 0.05). Compared with those in the EC group, the levels of LD, LDH, NO, and NOS in serum of the EP and ED groups were significantly lowered (P < 0.05) (Fig. 3B). Specifically, the levels of LD, LDH, NO and NOS in the EP group were lowered by 21.1% (P < 0.05), 6.5% (P < 0.05), 21.2% (P < 0.05) and 19.6%. Further, the levels of LD, LDH, NO and NOS in the ED group were lowered by 26.9% (P < 0.05), 6.5% (P < 0.05), 23.4% (P < 0.05) and 27.0% (P < 0.05).

Compared with those in the NC group, the levels of CK, MDA, and GSH-PX in the serum of the other groups were significantly increased (P < 0.05); compared with the EC group, the levels of CK, MDA, and GSH-PX in the serum of the EP and ED groups increased significantly (P < 0.05) (Fig. 3C). Specifically, the levels of CK and MDA in the EP group decreased by 13.3%, 45.4% (P < 0.05) and 20.8% (P < 0.05); and the levels of CK and MDA in the ED group decreased by 13.7% (P < 0.05), 51.3% (P < 0.05) and 21.2% (P < 0.05), respectively. The level of GSHPX in the EP group was 20.8% (P < 0.05), and the level of GSH-PX in the ED group was 21.2% (P < 0.05) higher than that in the EC group.

3.3.3. The biochemical parameters in liver, brain, and muscle tissues

Compared with those in the NC group, the levels of LD, LDH, NO, NOS, PA, CK, and MDA in the tissues of the other groups were significantly increased (P < 0.05). Compared with the EC group, the levels of LD, LDH, NO, NOS, PA, CK, and MDA in the EP and ED groups were significantly lowered (P < 0.05) (Fig. 4A; Fig. 4B; Fig. 4C; Fig. 4D; Fig. 4E; Fig. 4F; and Fig. 4J).

In comparison to those in the EC group, the levels of LD, LDH, NO, NOS, PA, CK and MDA decreased by 18%, 13% (P < 0.05), 22% (P < 0.05), 12.1% (P < 0.05), 8.8%, 12.4% (P < 0.05), and 21.8% (P < 0.05) in the liver of the EP group, respectively; decreased by 7.5% (P < 0.05), 9.8% (P < 0.05), 38% (P < 0.05), 26.7% (P < 0.05), 15%, 8.3% (P < 0.05) and 34.6% (P < 0.05) in the brain of the EP group, respectively; and decreased by 9.4%, 8.5% (P < 0.05), 23% (P < 0.05), 15.5% (P < 0.05), 20%, 11.4% (P < 0.05), and 20% (P < 0.05)in the muscle of EP group, respectively

In comparison to those in the EC group, the levels of LD, LDH, NO, NOS, PA, CK and MDA decreased by 14.8% (P < 0.05), 14.1%, 19.5% (P < 0.05), 16.7% (P < 0.05), 8.8%, 19.4% (P < 0.05) and 22.8% (P < 0.05)in the liver of the ED group, respectively; decreased by 7.5% (P < 0.05), 10.9% (P < 0.05), 38.1% (P < 0.05), 28.9% (P < 0.05), 15%, 16.6% (P < 0.05) and 32.2% (P < 0.05) in the brain of the ED group, respectively; and decreased by 10.6% (P < 0.05), 8.14% (P < 0.05), 23.1% (P < 0.05), 20.2% (P < 0.05), 13.3%, 14.4% (P < 0.05) and 18.6% (P < 0.05) in the muscle of ED group, respectively.

Compared with those in the NC group, the levels of GSH-PX, T-SOD, and CAT in the tissues of the other groups were significantly lowered (P < 0.05). Compared with group EC, the levels of GSH-PX, T-SOD, and CAT in tissues of the EP and ED groups were significantly increased (P < 0.05) (Fig. 4G; Fig. 4H; Fig. 4I).

Fig. 4. The level of LD(Fig. 4A), LDH(Fig. 4B), NO (Fig. 4C), NOS(Fig. 4D), PA(Fig. 4E), CK(Fig. 4F), GSH-PX(Fig. 4G), T-SOD(Fig. 4H), CAT(Fig. 4I), MDA (Fig. 4J) in liver, brain, and muscle. The content of liver glycogen, muscle glycogen, and ATP in tissues (Fig. 4K; Fig. 4L). Each group represents the mean ± SD. *P < 0.05, VS NC group; **P < 0.01, VS NC group. #P < 0.05, VS EC group; ##P < 0.05, VS EC group. Abbreviations: LD: Lactic Acid; LDH: Lactate Dehydrogenase; NO: Nitric Oxide; NOS: Nitric Oxide Synthase; PA: Pyruvate; CK: Creatine Kinase; GSH-PX: Glutathione Peroxidase; T-SOD: the Total Superoxide Dismutase; CAT: Catalase; MDA: Malonaldehyde; HG: Hepatic Glycogen; MG: Muscle Glycogen. NC: Normoxia Control Group; HC: Hypoxia Control Group; EC: Exhaustive Swimming Control Group; EP: Exhaustive Swimming Positive Group; ED: Exhaustive Swimming Drug Group.

In comparison to those in the EC group, the levels of GSH-PX, T-SOD and CAT increased by 15.9% (P < 0.05), 21.6% (P < 0.05) and 24.4% (P < 0.05)in the liver of the EP group, respectively; increased by 13.3% (P < 0.05), 13.8% (P < 0.05), and 9.8% (P < 0.05)in the brain of the EP group, respectively; and decreased by 12.1% (P < 0.05), 21.1% (P < 0.05), and 13.1% (P < 0.05) in muscle tissue of the EP group, respectively.

In comparison to those in the EC group, the levels of GSH-PX, T-SOD and CAT increased by 15.3% (P < 0.05), 33.8% (P < 0.05) and 24.8% (P < 0.05)in the liver of the ED group, respectively; increased by 13.6% (P < 0.05), 11.4% (P < 0.05), and 8.6% (P < 0.05) in the brain of the ED group, respectively; and increased by 15.4% (P < 0.05), 23.4% (P < 0.05), and 12.9% (P < 0.05) in muscle tissue of the ED group, respectively.

3.3.4. Detection results of energy substances in liver, brain, and muscle tissues

Compared with those in the NC group, the ATP, liver glycogen, and muscle glycogen levels in the other groups were significantly reduced (P < 0.05). Compared with those in the EC group, the ATP, liver glycogen, and muscle glycogen levels in the EP and ED groups were significantly increased (P < 0.05). The results are shown in Fig. 4K and L.

In comparison to those in the EC group, the levels of ATP (brain), ATP (liver), liver glycogen and muscle glycogen in the EP group increased by 180.1% (P < 0.05), 72.5%, 68.6% (P < 0.01) and 11.1% (P < 0.05), respectively, and the levels of ATP (brain), ATP (liver), liver glycogen and muscle glycogen in the ED group increased by 175.2%, 84.3%, 58.2% (P < 0.01) and 11.1% (P < 0.05), respectively.

3.3.5. Assessing the expression of related proteins in brain and liver tissues by Western blot

The expression levels of Bax/Bcl-2 and Nox2 in the liver tissue of the other groups were increased (P < 0.05) when compared with those in the NC group. The expression levels of Bax/Bcl-2 and Nox2 in liver tissue of the EP and ED groups were significantly reduced (P < 0.05) when compared with those in the EC group (Fig. 5a).

In comparison to those in the EC group, the expression levels of Bax/Bcl-2 and Nox2 in the liver tissue of the EP group were reduced by 3.1% (P < 0.05) and 17.5% (P < 0.05), respectively. In comparison to those in the EC group, the expression levels of Bax/Bcl-2 and Nox2 in the liver tissue of the ED group were reduced by 5.1% (P < 0.05) and 12.9%, respectively.

The expression levels of Bax/Bcl-2, Nox2, and Ampk in the brains of the other groups were improved significantly compared with those in the NC group (P < 0.05), and they were significantly reduced in the EP and ED groups compared with those in the EC group (P < 0.05) (Fig. 5b). In comparison to those in the EC group, the expression levels of Bax/ Bcl-2, Nox2, and Ampk in brain tissue of the EP group decreased by 16.7% (P < 0.01), 9.9% (P < 0.01), and 13.3%, respectively.

Fig. 5. (a) The expression level of Bax/Bcl-2 and Nox2 in liver tissue; (b) The expression level of Bax/Bcl-2, Nox2, and Ampk in brain tissue. Note: (A) NC: Normoxia Control Group; (B) HC: Hypoxia Control Group; (C) EC: Exhaustive Swimming Control Group; (D) EP: Exhaustive Swimming Positive Group; (E) ED: Exhaustive Swimming Drug Group.

In comparison to those in the EC group, the expression levels of Bax/Bcl-2, Nox2 and Ampk in brain tissue of the ED group decreased by 12.7% (P < 0.05), 4.8% (P < 0.01) and 17.5% (P < 0.05), respectively

3.3.6. Histopathological changes in rats

We clearly observed pathological changes in liver tissue by microscopy (Fig. 6). The morphological structure of the liver tissue was intact in the NC group, and the cells around the central vein were closely aligned. No necrotic cells were present, and there were no other pathological changes. In the HC group, the arrangement of the cells around the central vein of the liver was slightly loose, and the cell morphology was normal. In the EC group, the central venous structure of the rats was seriously disordered, the arrangement of cells was loosened, the cell volume was increased, there was edema between the cells, and cell necrosis was obvious. Compared with the EC group, the pathological results of the liver in the ED and EP groups were significantly improved.

Pathological sections of the brains of rats are shown in Fig. 7. In the NC group, the morphological structure of the brain was normal, the cytoarchitecture was clear, and the nucleus was obvious. In the HC group, there was some edema in the interstitium of the brain tissue. In the EC group, interstitial edema of the brain was obvious, and blood vessels exhibited edema and congestion. Compared with the EC group, the pathological results of the brain in the ED and EP groups were significantly improved.

Pathological sections of muscle tissue of rats are shown in Fig. 8. In the NC group, the morphological structure of muscle tissue was normal, the cytoarchitecture was clear, the nuclei were dispersed and arranged neatly, the band structure was ordered, and there was no inflammatory cell infiltration. In the HC group, accumulation of muscular nuclei appeared, and the cells were swollen. In the EC group, there was an obvious accumulation of muscular nuclei, the fascicle bands were nebulous, the bands were broken with large gaps, and tissue edema was obvious with inflammatory cell infiltration. Compared with the EC group, the pathological results of muscle tissue in the ED and EP groups were significantly improved.

4. Discussion

Oxygen is an important factor in maintaining the normal life of organisms. Hypoxia leads to tissue hypoxia and causes abnormal changes in the body that seriously affect health. Mild hypoxia can cause the body to breathe deeper and accelerate breathing, increasing cardiac output. At the same time, some compensatory changes occur in the blood to ensure the blood supply of the related organs of the organism. When severe hypoxia occurs, compensatory changes in the body cannot or do not occur completely. It easily causes abnormal metabolism of the body and even leads to death.

Exercise fatigue is a complex physiological process that is produced by the organism exercised at a certain intensity or for a certain period of time and is mainly referred to as experiencing the high intensity and high load activities. The motor capacity of the body is greatly reduced, and physical strength and mental power show a degree of damage and cannot maintain or withstand the predetermined intensity of exercise. When people enter the plateau from the plain area, exposure to a highaltitude hypoxic environment causes metabolic disorders and decreases body memory, cognitive ability, and work efficiency, easily causing exercise-induced fatigue (Jiang et al., 2013)

At present, there are many studies about the mechanism of sports fatigue; the mechanism of sports fatigue mainly includes exhaustion of energy substances, accumulation of bad metabolites, imbalance of the internal environment, and high levels of free radicals (Carter, 2014).

However, the mechanism of sports fatigue under high altitude is relatively complicated, in which hypoxia and free radical damage caused by exercise are the direct causes. In the model animals subjected to overload exercise under hypoxic conditions, BUN, CRE, UA, PA, LD, and other metabolites accumulated, the levels of free radicals NO and MDA increased significantly, and the contents of liver glycogen and muscle glycogen decreased. PK and LDH are the key enzymes in glycolysis, and CAT, GSH-PX, NOS, and T-SOD are mainly involved in lipid peroxidation in vivo and affect the production of free radicals.

Cistanche phenylethanoid glycosides have an Anti-fatigue function

It is generally believed that the mechanism of sports fatigue mainly includes insufficient energy supply, accumulation of metabolites, and excessive production of free radicals. BUN, CRE, UA, PA, and LD are metabolites produced by the body, and excessive accumulation of these metabolites can have an effect on the organism (Hong et al., 2015; Huang, Huang, Ye, & Qin, 2010; Li et al., 2016). BUN is the metabolic product of proteolysis, and its content increases with increasing exercise load and reflects the exercise endurance of the body. LD is a main aerobic glycolysis product that is converted from PA to LD under the action of LDH, and accumulating LD in the body is likely to cause fatigue (Chi et al., 2015; H.-p.; Zhao et al., 2017). This study found that an equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides can significantly reduce the levels of BUN, CRE, UA, PA, and LD and can significantly delay the onset of movement fatigue.

NO and MDA are two kinds of free radicals in the body. When the organism is prone to produce excessive free radicals during hypoxia, the high free radical content can lead to lipid peroxidation and cell trauma (Nam, Kim, & Jeong, 2016). We found that an equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides can significantly decrease NO and MDA, inhibit the production of too many free radicals and protect cells from damage.

ATP, liver glycogen, and muscle glycogen, as energy materials, play important pathophysiological roles and can provide energy for the body (Y. Chen et al., 2016; Lee et al., 2015). When body energy is insufficient, liver glycogen and muscle glycogen provide energy for the body through gluconeogenesis. Our research group found that an equal proportion of a mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides can significantly increase the levels of ATP, liver glycogen, and muscle glycogen and delay the onset of movement fatigue.

The body has many enzymes involved in energy supply, free radical scavenging, and metabolite regulation. These enzymes also reflect the energy metabolism of the body. PK and LDH are the key enzymes in glycolysis. CAT, GSH-PX, NOS, and T-SOD are mainly involved in lipid peroxidation and affect the production of free radicals (Ding et al., 2011; Kumar, Anand, Singsit, Khanum, & Anilakumar, 2013; Nam, Kim, & Jeong, 2016; Ni et al., 2013; Ramesh et al., 2012; Wang et al., 2010; M.; Zhao, Regenstein, & Ren, 2011). This study found that an equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides can significantly reduce the levels of PK, LDH, and NOS and can increase the levels of CAT, GSH-PX, and T-SOD, protect tissue cells from damage and improve exercise tolerance under hypoxic conditions.

Bcl-2 and Bax are two important proteins in the process of apoptosis, in which Bax is a protein that promotes apoptosis, while Bcl-2 is a protein that inhibits apoptosis. The expression ratio of the two proteins (Bax/Bcl-2) is of great significance to apoptosis (Jia et al., 2013; Miao et al., 2013). Caspase-3 is also an important apoptotic protein that is a key factor in the process of apoptosis. DNA cleavage factor is activated, and endonuclease nucleic acid is activated, which eventually leads to cell death. This process plays a vital role in the process of apoptosis (Choudhary, Al-Harbi, & Almasan, 2015). Ampk is an AMP-dependent protein kinase that is closely related to the regulation of energy metabolism and plays an important role in maintaining glucose balance. After a lot of exercises, Ampk in the body is activated (Niederberger, King, Russe, & Geisslinger, 2015). Nox2 plays an important role in inflammatory reactions and oxidative stress and is the main source of ROS. Nox2 can transmit electrons through intracellular NADPH and permits extracellular oxygen to enter through the cell membrane, which will eventually lead to the production of superoxide (Khayrullina, Bermudez, & Byrnes, 2015). In this study, it was found that a mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides could reduce the expression of apoptotic proteins and reduce Ampk and Nox2 in hypoxic exhaustive swimming rats. Therefore, the anti-fatigue effect of the mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides may be related to delaying apoptosis

In summary, an equal proportion mixture of gardenia yellow pigment and Cistanche phenylethanoid glycosides has anti-hypoxia and anti-fatigue effects, and the related mechanisms need to be further studied.

Cistanche phenylethanoid glycosides have an Anti-fatigue function

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