Dietary cucurbitacin E reduces high-strength altitude training induced oxidative stress, inﬂ ammation and immunosuppression

: Professional athletes conduct high-intensitive hypoxic training often accompanied by the increase of many infl ammatory-related cytokines and immunosuppression. Cucurbitacin E (CucE), as a triterpenoid isolated from Cucurbitaceae plants, exert potential anti-cancer and anti-infl ammatory. However, it is unknown whether that the CucE could be used as dietary supplement for athletes to improve infl ammatory response and immunosuppression. In this study, we established the simulative hypoxic training rat and monkey models and evaluated the effects of CucE on immune- and infl ammation-related factors. Obvious improvement on pro-infl ammatory factors and pro-lymphocyte proliferation activities were showed in CucE treated rats compared with the control. Further supplement of CucE in professional meals for cynomolgus monkeys with 4-weeks high-intensitive hypoxic training also exert effects on altitude-induced oxidative stress, infl ammation and immunologic function. Furtherly, we explored the underlying mechanism of CucE in human Jurkat T cells and results showed that CucE may exhibit immunosuppressive effect by attenuating critical cytokine expression through down-regulating the NF-κB signaling pathway. In conclusion, CucE is expected to be a potential dietary supplement for athletes to ameliorate the infl ammation and immunosuppression caused by high-intensitive exercise.


INTRODUCTION
Athletes in several sports, such as basketball, need various modalities of hypoxic training to enhance their capacity of blood oxygen carrying for the better energy and stamina, and ultimately improve sports performance (Akpinar 2016, Bonci 2011, Ploszczyca et al. 2018. Up to now, many elite athletes and sports teams have often utilized high-strength hypoxic training for the preparation of different levels of competition, although, its efficacies remains controversial from a research perspective (Franchini et al. 2011, Hamlin et al. 2019. However, while the combined strict hypoxia and high-intensitive exercise brings the obvious improvement on athletic abilities for athletes, it also introduces the enormous risks on immune system, including immunological disturbances, infections and illness (Blume et al. 2018, Canis & Santos 2019, Doherty et al. 2019, Walsh 2018. Not only that, the obvious increase of systemic infl ammatory cytokines following high-intensitive altitude training indicating a relatively unambiguous immune system response (Khanna et al. 2018, Shen et al. 2012). In addition, altitude training were also reportedly related to the subsequent increase in production of the free radical and oxidative stress (Quindry et al. 2016, Sinha et al. 2009).
According to several reported traditional ethno-biomedical researches (Oikawa et al. 2012, Ozkan et al. 2016, Zhao et al. 2016, plants have been used to treat various human diseases including excessive oxidative stress and inflammation. Among numerous functional plants, cucurbits were demonstrated the abilities of immunoregulatory, antiinflammatory and liver protection. Several bioactive compounds from cucurbits family were early recognized with main biological values in both in vivo and in vitro assays. Cucurbitacins are a group of tetracyclic triterpenes isolated from Cu cucurbitaceae plants possessing extensive pharmacological properties (Chen et al. 2012, Hussain et al. 2019. Cucurbitacin E (CucE), one of the abundant members of the cucurbitacin family, has similar pharmacological activities to other cucurbitacins. According to previous researches, the anti-cancer activity of CucE has been proposed to be mediated by inhibiting signal transducer and activator of transcription 3 phosphorylation in cancer cells (Jevtic et al. 2016, Murtaza et al. 2017. Beyond the reported anti-cancer activities, CucE has achieved more and more attention for its anti-inflammatory and immunoregulatory activities (Shi et al. 2015). However, whether the CucE could use as an effective dietary supplement to ameliorate the high-strength altitude training induced oxidative stress, inflammation and immunosuppression is largely unknown.
Thus, the aim of current study was conducted to determine whether the administration of CucE influences systemic oxidative stress and inflammation related biomarkers in response to the high-strength altitude training in rat and cynomolgus monkey models. We also aimed to explore the action mechanism of CucE on suppression of inflammatory response and immune abnormality in the levels of mRNA and protein expression in human Jurkat T cells.

Materials
Cucurbitacin E, as a kind of Chinese herb extracts and dietary supplement, was obtained from Shanghai Aibo Biotechnology Co., Ltd (Shanghai, China). Mouse, rat and human lymphocytes were purchased from Shanghai Yan sheng Biotechnology Co., Ltd (Shanghai, China). Wistar rats and cynomolgus monkeys were obtained from Guang Dong Experimental Animal Center and were allowed with free access to diet and water, and acclimated to controlled ambient conditions of 12-h light/dark cycle before establishment of hypoxia training model. Jurkat T cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Animal studies were approved by the Institutional Animal Care and Use Committee of the Soochow University, and performed according to the guidelines.

Establishment of rats and cynomolgus monkeys hypoxia training model
In the present study, the 4-6 weeks old SD rats, weighting 200-250 g, were subjected to weightbearing swimming training by hanging a 10 g lead pellet in the lower abdomen and placing it in the Morris water maze simulating at 4000m. The rat's mouth and nose were submerged in water for 7 s, and then picked up and dried quickly. Training time interval: 1 time/d, 6 times/ week, rest interval 1 d, total training 4 weeks. The experimental rats were randomly divided into 3 groups (n=6): (1) normal ones without hypoxia training. The rats with altitude hypoxia training orally treated (2) with CucE (1mg/kg) or (3) without CucE for 4 weeks.
High-strength altitude training of cynomolgus monkeys in the current study was performed utilizing altitude training model in primary way of previous reports (Shi et al. 2015).
Dietary supplementation with cucurbitacin E for cynomolgus monkey was carried out as follows: half amounts of cynomolgus monkeys were allocated to dietary supplementation with cucurbitacin E (50 μg/per day) whereas the other half ones were allocated to the control food treatment. Dietary supplementation with cucurbitacin E was provided twice a week.

Lymphocyte transforming assay
Rat serum and human serum were collected before and at week 2, 4, 5 (a week rest period) and incubated with mouse, human lymphocytes and mouse, rat lymphocytes, respectively. Iymphocyte transformation indexes were measured with previous reported MTT method (Wei et al. 2003).

Determination serum levels of cytokines and immunoglobulin
Quantitative determination of serum levels of IL-2, IL-6, IL-8, IL-10, IL-1β and TNF-α were conducted using enzyme-linked immunosorbent assay (ELISA) kit, which were purchased from R&D Systems (Minnesota, America). Serum immunoglobulin levels (including IgA, IgG, and IgM) were detected using Abbott Laboratories automatic biochemical analyzer (Chicago, America). All the operations were conducted according to the user protocol.

Cell culture and detection
Jurkat T cells were cultured in RPMI-1640 medium with 2mM, 10% FBS, 50 μg/mL streptomycin, and 50 U/mL penicillin at 37°C. Cytotoxicity assays were conducted without or with different concentration of CucE under the stimulation of PDB (0.5 μM) plus Ion (0.5 μg/mL). Proliferation of cells were quantified according to the measurement of the absorbance at 450 nm by using a Bio-Rad 680 microplate reader (Hercules, America).

Detection of PBMC activated surface molecule
The peripheral blood mononuclear cells (PBMCs) of volunteers was separated by density gradient centrifugation, and then were cultured without or with different concentration of CucE (1, 3, 10 μM) under or not under the stimulation of PDB (0.5 μM) plus Ion (0.5 μg/mL). The PBMCs were washed by using PBS (0.1% NaN 3 and 2% fetal calf serum) and fixed by 4% paraformaldehyde. The further detection of PBMC activated surface molecule (CD25 and CD69) were conducted using the FACSCalibur flow cytometer (BD, America) after 24h culture.

Western blot analysis and RT-PCR analysis
Western blot analysis were conducted to evaluate the Jurkat T cell lystate as described previously (Wang et al. 2015). All the antibodies used in these detection were obtained from Cell Signaling Technology (Boston, America). Each band of gel was quantified by Quantity One® 1-D software (Bio-Rad, America). Total RNA were obtained from Jurtkat T cells, and subsequently transcribed into cDNA by using PrimeScript RT kit (Thermo Fisher, America). The primers used in this PCR and relative quantification of gene expression were conducted as previously described (Wang et al. 2015).

Data analysis
All measured variables are presented as mean ± SD. Differences in all parameters were tested using one-way ANOVA. P values lower than 0.05 were considered significant.

Effects of CucE on antioxidant capacity, oxidative stress and inflammation biomarkers in rat model of stimulating altitude training
In this study, the rats in stimulating altitude training group were trained by means of swimming for 4 consecutive weeks at 4000 m followed by reoxygen training for 1 week. As a result, the rat model of altitude training were successfully established. Rats in the normal control group 1ived and trained in normal conditions, and those in the other groups all trained with swimming in the stimulating altitude environment for 4 weeks. During the training and CucE administration period, body weight changes of rats in the stimulating acute hypoxia training groups (treated with CucE or not) and normal condition group were showed in Figure 1. The body masses in all the 3 groups were positively increasing, whereas the growth rate of weight in hypoxia training group (without CucE) was lowest indicating that the CucE treatment exert improvement on the normal weight gain. Changes in serum pro-inflammatory factors were furtherly detected and showed in Figure  2. Except for the IL-10, plasma levels of IL-6, IL-8, IL-2, IL-1β and TNF-α in altitude training model rats were all significantly increased after 4-week experiment period, whereas the rats treated with CucE effectively reverse abnormality of these cytokines indicating the potential anti-inflammatory effects of CucE in highintensitive training individuals.
Effects of serum from altitude training rats on mice lymphocyte transformation Furthermore, we evaluated the effects of the serum collecting from cynomolgus monkeys before, at week 2, 4 (after hypoxia training) and week 5 (after a week rest period) on mouse or rat lymphocyte proliferation. As is showed in Figure 3a, serum from normal humans (without hypoxia training) exert stable activity to promote mouse or rat lymphocyte proliferation, whereas the serum in two hypoxia training group (without CucE) showed obvious inhibition effects on lymphocyte proliferation indicating that the high-intensity training may induced immunosuppression. In addition, administration of CucE in cynomolgus monkeys significantly improve the immunosuppression and help to rapidly return to a normal baseline after a week rest period.

Effects of CucE on altitude-induced oxidative stress, inflammation and immunologic function in cynomolgus monkeys
As is illustrated in Figure 4, plasma levels of IL-6, IL-8 and TNF-α of cynomolgus monkeys were significantly increased (P<0.05), and the plasma level of IL-10 was obviously decreased abnormality after four-week hypoxia training, whereas the administration of CucE effectively reduce the abnormality of these cytokines. Above results indicated that there are inflammation reaction exists in cynomolgus monkeys after high-intensitive hypoxia training, and CucE could improve the training induced inflammation.
As shown in Figure 5, serum level of IgG and IgA were gradually decreased in cynomolgus monkeys during the four-week altitude training period, and administration of CucE could obviously reverse these changes.
According to previous reports (Hui et al. 2019, Vajapey et al. 2014), many free radicals were caused by high-intensitive training and accumulated due to weaker scavenging capacity of anti-oxidation enzyme system in athletes. Then the extensive cellular damage comes with the inhibition of T and B lymphocyte cells. In this study, cell ratio and quantity of B cells, T cells and CD3 + CD4 + T cells were all significantly decreased after 4-weeks altitude training, whereas the CucE treatment kept the lymphocyte subsets in a relatively stable state. In addition, CD3 + CD4 + T cells were nor obviously changed in both cynomolgus monkey groups with or without CucE (Table I).
Effects of serum from altitude training cynomolgus monkeys on mice lymphocyte transformation Furthermore, we evaluated the effects of the serum collecting from cynomolgus monkeys before, at week 2, 4 (after hypoxia training) and week 5 (after a week rest period) on mouse or rat lymphocyte proliferation. As is showed in Figure 6, serum from normal cynomolgus monkeys (without hypoxia training) exert stable activity to promote mouse or rat lymphocyte proliferation, whereas the serum in two hypoxia training group (without CucE) showed obvious inhibition effects on lymphocyte proliferation indicating that the high-intensity training may induced immunosuppression. In addition, administration of CucE in cynomolgus monkeys significantly improve the immunosuppression and help to rapidly return to a normal baseline after a week rest period.

Mechanism of potential immunomodulatory action of cucurbitacin E
In this study, we chose the PDB+Ion activated Jurkat T cell as the model to initially evaluate the potential immunomodulatory action and mechanism of CucE. As is showed in Figure  7a, there is no significant inhibition of CucE within 12 hours incubation on cell proliferation when its concentration was 10 μM or less in WST-1 test. When the incubation time was extended to 24 hours, the cell viability was more than 90% (10μM) indicating that the cytotoxicity of CucE incubation was limited at the relatively low concentration (10μM), and the doses of CucE used in the subsequent in vitro assays were determined to 1, 3, and 10 μM. In addition, human PBMCs, which were activated by incubation of PDB (0.5 μmol/L) and Ion (0.5 μg/ ml) were stimulated in the presence or absence of CucE for consecutive 48 h. Expression of the CD69 and CD25 within CD3 + T cells were assessed using FACS method, and results showed that CueE treatment exerts both inhibiting effect on CD25 and CD69. Activated T cells secret some cytokines, such as IL-2 and TNF-α, play very important roles in immune system response. We furtherly investigated effects of CucE treatment on the transcript levels of IL-2, TNF-α and IFN-γ.
Quantitative RT-PCR results proved that the treatment of PDB+Ion significantly increased the mRNA levels of above cytokines, and the CucE incubation exert obvious down-regulatory effect in a dose-dependent manner (Figure 7c). Similar inhibition effects on protein levels of above cytokines were also showed in Figure 7d and suggested that incubation of CucE attenuate the immune response of T cells by inhibiting the cytokines both in mRNA and protein levels.
As CucE significantly decreased both mRNA and protein levels of IFN-γ, IL-2 and TNF-α, and NF-κB pathways are crucial in modulating the transcription, expression of several immune system related cytokines according to the previous reports (Hsu et al. 2016, Yao et al. 2014. Therefore, we conducted subsequent experiment to evaluate the effects of CucE on the NF-κB signaling in PDB+Ion activated Jurkat T cells. As shown in Figure 8, analysis of western blot revealed that treatment of CucE obviously reduced the phosphorylation levels of p65 and IκB which were significantly stimulated by incubation of PDB+Ion. We furtherly examined whether CucE treatment could affect the NF-κB/ p65 nuclear accumulation. Analysis of western blot showed that the stimulation of PDB+Ion induced a significant nuclear accumulation in pre-treated Jurkat T cells, whereas the treatment of CucE decreased the NF-κB/p65 level in the nucleus.
Above results indicate that the CucE down-regulates the NF-κB signal pathway via inhibiting phosphorylation of NF-κB and the nuclear translocation of NF-κB/p65.

DISCUSSION
In order to improve the blood oxygen capacity and endurance for the better sports performance, athletes of basketball and other similar sports often need hypoxic training under different intensity. However, the often utilized high-strength hypoxic training brings not only the significant improvement on athletic abilities, but also induces the enormous risks on immune system, such as hypoimmunity and immunological disturbances.
Cucurbitacin are kinds of cytotoxic triterpenoid substances which are originally isolated from Cucurbitaceae plants. Several cognates of cucurbitacin were previous identified (Hunsakunachai et al. 2019, Lui et al. 2009), and their pharmacological efficacies had also been reported, such as anti-fertility, purgative and anti-tumor activities. Previous researches revealed that CucE, as a promising compound with wide spectrum of pharmacological properties, possesses enormous anti-tumor and anti-Inflammation potential (Hussain et al. 2019, Jevtic et al. 2016, Murtaza et al. 2017.
In this study, we investigate the immunomodulatory effects of CucE on hypoxia training rat model, and carefully explored the potential related mechanism in human Jurkat T cells. In addition, the potential application of CucE for basketball athletes, who needs to often conduct hypoxic training, as a promising agent in modulating the adaptive immune response and alleviate athletes' elevated inflammatory cytokines were also evaluated in cynomolgus monkeys, the closest animal model to humans.
The weight change in this study mean a reflection of physical status of animals. As shown in Figure 1, the high-strength altitude training reduced the body weight without any supplement while the dietary CucE effectively reversed this trend indicating the protective effects of CucE on the integral physical status of rats.
Recent studies found cytokines play important roles in multiple physiological behaviors, including the cells proliferation, immune function and repairing effects of damaged tissues. The effects of high intensity exercises on cytokines have been mentioned in many literatures, such as one-time strenuous exercise leads to a more than 100-fold increase in IL-6 level. However, there are few reports on the effects of long-term (over 2 weeks) intensive training on serum cytokines of animal models, especially for primates. Therefore, in this study, we investigated the effects of high-intensity and low-oxygen training on the changes of cytokines in both rat and primate models, as well as the biological effects of CucE. Results in Figures 2 and 4 showed that IL-6, IL-8 and TNFalpha increased and IL-10 decreased indicating an abnormal state immune system of rat and cynomolgus monkey models after 4-weeks intensive training, whereas the administration of CucE efficiently improved these changes in both two species.
Previous reports have shown that certain immunosuppressive substances can be produced in the serum of athletes after high-intensitive exercise (Pedersen et al. 2011, Sato et al. 2016. In this study, we also found that incubating human and mouse lymphocytes with stress serum of rats (after 4-weeks altitude training) can significantly reduce transformation efficiencies, and serum from cynomolgus monkeys also has similar effects on the transformation of rat and mouse lymphocytes. Another similar biological phenomenon were also showed in the serum from CueE treated groups which exert a significantly reduced inhibition of lymphocyte transformation, suggesting that CucE, as a food or some other forms of supplement, has great potential to improve the immune suppression caused by excessive exercise.
To further explore our hypothesis, we investigated the effects of CucE administration on serum concentration of immunoglobulin after high-intensitive exercises. Immunoglobulin, as the glycoprotein secretory by the mature plasma cells differentiated from B cells in response to stimulation of antigen, can directly against the corresponding pathogenic microorganism and toxins, and induce complement activation, phagocytosis, and so on. Immunoglobulin is main composition of the humoral immune and certainly related to health and fatigue status. Our results in Figure  5 showed that 4-weeks high-intensity training in cynomolgus monkeys led to a significant decrease in serum level of IgG, which was more sensitive than IgA and IgM, suggesting that humoral immune function of cynomolgus monkeys decreased after excessive exercise. Administration of CucE also could effectively improve the sharp decrease of IgG.
Percentage and count of NK cell, B cells, T cells and CD3 + CD4 + T cells were all decreased significantly in the peripheral blood from cynomolgus monkeys after 4-weeks high-intensitive training. Under normal circumstances, the ratio of CD3 + CD4 + / CD3 + CD8 + is in a relatively balanced state which keeping the immune function also is stable. The decrease of CD4 + /CD8 + ratio is a manifestation of cellular immunosuppression, which may cause immune dysfunction or immune diseases. According to previous reports (Campbell et al. 2012, McLeod and He 2010, Pinto et al. 2014, we suspect that the to many high-intensitive exercises cause too much free radicals accumulation leading extensive damage to immune cells, thus inhibiting the function of T lymphocytes and B lymphocytes. Meanwhile, the levels of B cells and Th cells were significantly decreased. Thus, we speculated that the inhibition of B cells and Th cells might be the main reason for the significant decrease of IgG in cynomolgus monkeys after high intensity exercises.
In the above studies, we found that intake of CucE can effectively reduce the occurrence of immunosuppression and inflammatory response in cynomolgus monkeys after high-intensity exercise. In order to explain and explore the regulatory effect of CucE on adaptive immune function and its possible mechanism, as well as the potential of using CucE as an anti-inflammatory drug or a dietary supplement to alleviate the increase of pro-inflammatory cytokines in athletes.
As shown in Figure 7, CucE significantly suppressed the expression of CD69 and CD25 on the surfaces of CD3 + T cells in PBMC stimulated with PDB plus Ion. PDB+Ion induced expression of IL-2, TNF-α and IFN-γ were dose-dependently inhibited by CucE. The mRNA levels of these cytokines in activated Jurkat T cells were also decreased upon CucE treatment. CucE decreased the phosphorylation levels of inhibitor of κB (IκB) and NF-κB/p65 in PDB+Ion stimulated cells. The nuclear translocation of NF-κB/p65 was also significantly suppressed in the presence of CucE. The phosphorylation of p38 MAPK, JNK and Erk1/2 was not decreased by CucE treatment.
The effects of CucE as a dietary supplement on oxidative stress, inflammation, and immunosuppression induced by excessive exercise were validated in rat and cynomolgus monkey models. The validation results of different species models are basically consistent, especially in non-human primates, indicating the application potential of this CucE.

CONCLUSION
Our study found that administration of CucE could efficiently inhibited the expression of some cytokines with negative effects, such as TNF-α, and down-regulated the NF-κB signaling pathway, suggesting that CucE has certain potential in regulating adaptive immune function and suppressing inflammatory response. In the follow-up study, we will investigate the longterm safety as well as acute toxicity of CucE administration under large dose. We will explore the combined administration of CueE with other dietary fiber and anti-inflammatory foods or as one of the additives in sports drinks for the better application prospect for alleviating athletic inflammation and immunosuppression in caused by regular high intensity training.