20(R)-ginsenoside Rg3, a product of high-ef ﬁ ciency thermal deglycosylation of ginsenoside Rd, exerts protective effects against scrotal heat-induced spermatogenic damage in mice

: Heat stress (HS) reaction can lead to serious physiological dysfunction associated with cardiovascular and various organ diseases. Ginsenoside Rg3 (G-Rg3) is a representative component of ginseng rare saponin and can protect against multiple organs, also used as functional food to adjust the balance of the human body, but the therapeutic effect and molecular mechanism of G-Rg3 on male diseases under HS are underexplored. The aim of the present study, G-Rg3 was prepared through the ef ﬁ cient conversion of ginsenoside Rd and investigate the contribution of G-Rg3 to testicular injury induced exposure to HS. All mice were divided into four groups as follows: normal group, HS group, and HS+G-Rg3 (5 and 10 mg/kg) groups. G-Rg3 was administered orally for 14 days, then exposed to a single scrotal heat treatment (43°C, 18min) on the 7th day. After HS treatment, the morphology of testis and epididymis changes, and caused a signi ﬁ cant loss of multinucleated giant cells, desquamation of germ cells in destructive seminiferous tubules, and degenerative Leydig cells, further destroying the production of sperm. After administration G-Rg3 (5 and 10 mg/kg/day) for 2 weeks, the spermatogenic-related indexes of testosterone levels and superoxide dismutase (SOD) activity, glutathione (GSH) content signi ﬁ cantly ( p < 0.01) increase compared with the HS group. Moreover, G-Rg3 treatment effectively ameliorated the production of malondialdehyde (MDA) ( p < 0.05 or p < 0.01). Importantly, G-Rg3 exhibited the protective potential against HS-induced injury not only suppressing the protein levels of heme oxygenase-1 (HO-1), hypoxia-inducible factor-1 α (HIF-1 α ), and heat shock protein 70 (HSP70) but also modulating the Bcl-2 family ( p < 0.01 or p < 0.001) and activation of mitogen-activated protein kinase (MAPK) signaling pathways ( p < 0.01). For most of the parameters tested, the HS+G-Rg3 (10 mg/kg) group exhibited potent effects compared with those exhibited by the low dose (5 mg/kg) group. In conclusion, the present study demonstrated that G-Rg3 exerted protective effects against HS-induced testicular dysfunction via inhibiting the MAPK-mediated oxidative stress and apoptosis in mice.

system (Agarwal et al., 2015).Increasing heat stress (HS) reactions are emerging and becoming more prevalent because of modern lifestyle, including mental stress, smoking, high temperature, and air pollution adverse factors (De Blois et al., 2015;Hughes and Acerini, 2008).Importantly, abundant evidence suggested that male reproductive dysfunction is closely related to environmental changes, affecting testicular quality and spermatogenesis significantly (Durairajanayagam et al., 2015).This might be due to high temperatures increase the testicular metabolism lack of adequate blood supply and result in local hypoxia damaging the testicular tissue (Bromfield et al., 2017;Lin et al., 2016), which is corroborated by studies in a number of animal models (Shen et al., 2019;Sui et al., 2019).Therefore, the correction or prevention of heat-induced sterility is a problem of major concern.
Early experimental evidence by Phuge, Hu, and Cai, indicated that scrotal temperature is essential to maintaining an optimal environment in testicular function (Cai et al., 2011;Hu et al., 2019;Phuge, 2017).Recent studies have shown that scrotal temperatures above the normal range caused oxidative stress and germ cell death in testes, leading to male infertility (Han et al., 2019;Zhang et al., 2012).Moreover, continuing HS also lead to severe damages on the testis, such as hypoxia in testes and endogenous or exogenous toxins (including peroxide anions and hydrogen peroxide), and related to the DNA damage and repair (Paul et al., 2008), and the production of hypoxia-inducible factor-1 (HIF-1) in the male germline.
HS reaction due to elevated temperatures involved in the activation of the transcription factor, such as HIF-1 (Baird et al., 2006), also including the production of free radicals and reactive oxygen species (ROS) (Costa et al., 2018).Spermatozoa are extremely sensitive to ROS-induced damage (Aitken and Clarkson, 1987).Meantime, overproduction of ROS can induce spermatogenesis-related negative changes, such as sperm capacitation and acrosome reaction (Castillo et al., 2019;Stival et al., 2016).As spermatogenesis is susceptible to oxidative stress, and peroxidative injury is considered to be one of the important reasons for impaired testicular function (Houston et al., 2018;Li et al., 2014).Oxidative stress occurs also related to the disturbance of the antioxidant defense systems, such as the changes of lipid peroxidation malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) (Kim et al., 2013a).Meanwhile, heme oxygenase-1 (HO-1) and heat shock protein 70 (HSP70), play a potential target in heat stress-induced spermatogenic cell disorders (Stahli et al., 2019).Among heat shock proteins (HSPs) are a group of stress-responsive proteins expressed widely in various organs, and also vitally important in inhibiting cellular apoptosis, keeping intracellular homeostasis of redox, and maintaining mitochondrial integrity (Kim et al., 2015).More importantly, oxidative stress could result in cell cycle arrest and trigger apoptosis, the delicate balance between oxidative stress (Niu et al., 2020) and the apoptotic pathway is critical during the process of spermatogenesis (Xu et al., 2019).Increasing evidence demonstrates that Bax, Bcl-2, and caspase family are involved in the regulation of apoptosis of germ cells and Leydig cells (Fischer and Schulze-Osthoff, 2005;Kheradmand et al., 2012).Despite such a severe negative impact of HS on spermatogenesis, research on the specific mechanism of heat-induced spermatogenic damage is not clarified.
Panax ginseng C. A. Meyer is a health-promoting traditional Chinese medicine with long medication history, its saponins (ginsenosides) were considered as the main active ingredient of P. ginseng (Jin et al., 2015).Especially, G-Rg3 is a representative rare saponin and the most important active component of P. ginseng processed products (Red or Black ginseng) (Kim et al., 2019;Zhang et al., 2020).Importantly, ginsenosides Rb1, Rb2, Rc, and Rd with sugar moieties attached to the β-OH at C-3 and/or C-20, the sugar moieties at C-3 or C-20 were deglycosylated and dehydrated by heat processing, and they were gradually changed into 20(S/R)-Rg3, Rk1, Rg5 (Lee et al., 2009) and 20(S/R)-Rh2 (Fig. 1).Modern pharmacology research showed G-Rg3 was effective for various diseases, such as anti-tumor, protects against multiple organ damage, promoting immune response, and numerous powerful pharmacological effects (Kim et al., 2020;Park et al., 2020).Further, P. ginseng has been traditionally used for therapeutic in boosting libido and treating infertility in men (Park et al., 2016).In recent years, increasing studies on chemical, pharmacological and clinical aspects have proved that the pharmacological action of G-Rg3 provides broad prospects of the application (Fan, 2019;Phi et al., 2019;Zhou et al., 2018).
Previous studies have indicated that ginsenoside Rd through efficient thermal deglycosylation to conversion rare ginsenosides, and improved anticancer activity (Kim et al., 2013b), but few studies have focused on the molecular mechanism through that rare ginsenosides treat a series of male diseases in vivo.Thus, in order to further develop natural products and better treat male diseases such as spermatogenic damage induced by HS.The present study aimed to use PPD-type ginsenoside Rd through hightemperature acid treatment to the purification of rare ginsenoside G-Rg3, and explored the effects and the available evidence on the role of G-Rg3 in HS-induced spermatogenic damage and provided its possible mechanism.

Sample preparation
G-Rg3 with a purity of 95.0% was the purification of rare ginsenoside from PPD-type ginsenoside Rd by formic acid treatment at 90°C.Specifically, take about 1.0 g of ginsenoside Rd powder, add 50 mL of 0.5% formic acid aqueous solution, and in a water bath at 90°C for 3 h by heat processing.The HPLC chromatogram of ginsenoside transformation products is illustrated in Fig. 2. The peak of ginsenoside Rd disappeared, and the peaks of ginsenoside 20 (S/R)-Rg3, Rk1, and Rg5, were newly detected in transformation products (Fig. 2B).The quantitative analysis of G-Rg3 was implemented on a Hypersil ODS2 column using high-performance liquid chromatography (Waters HPLC, Milford, MA, USA) at 203 nm.The detection method of HPLC, as previously described with a few adjustments (Xue et al., 2017).

Animals and experiments design
Eight-week-old ICR male mice, weighing 22-25 g, were obtained from YISI Experimental Animal Co., Ltd., with a Certificate of Quality No. SCXK (JI) 2016-0003 (Changchun, China).The mice underwent adaptive feeding for at least one week before the formal experiment.The mice were exposed to controlled temperature (22.0 ± 2.0°C) and humidity (60 ± 10%) in a 12 h light/dark pattern in plastic cages and a free supply of food and water.All experimental animal processing procedures were strictly performed according to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2016).All animals' protocols were in accordance with the Ethical Committee for Laboratory Animals of Jilin Agricultural University (Permit No.: ECLA-JLAU 2019-00718).
The mice were randomly divided into 4 groups: Normal (N) group (saline for 14 days without HS), HS group (scrotal hyperthermia; 43°C, 18 min), HS + G-Rg3 group (5 mg/kg), and HS + G-Rg3 group (10 mg/kg).Since there is currently no therapeutic agent for scrotal heat-induced spermatogenic damage in the clinic, a group of positive drugs was not set up in the present work.G-Rg3 were dissolved in sodium carboxymethyl cellulose (CMC-Na) to give different doses of 5 and 10 mg/kg according to our previous work and preexperiments (Zhou et al., 2018), respectively.Mice were administered G-Rg3 orally once daily for 14 days, on the 7th day, all animals except the N groups, other groups were exposed once to HS at 43°C for 18 min to establish a scrotal hyperthermia model as previously described (Leng et al., 2019).For HS treatment, the lower third of the body (hind legs, tail, and scrotum) was submerged in a mini water bath.After 30 min, each animal was dried and returned to its cage.7 days after HS exposure, the animals were kept in starvation and were euthanized under anesthesia.The testes were excised, weighed, and maintained at −80°C for the subsequent assays.

Determination of serum testosterone levels
Serum was separated from blood after centrifugation (3500 rpm, 10 min) twice at 4°C (Mi et al., 2019).The testosterone level in serum was detected in accordance with the instructions of the commercial kits.Briefly, this kit utilizes the antibodies specific, as well as HRP-conjugated streptavidin solution, to estimate spermatogenic-related products.The absorbance was taken at 450 nm using a MultiskantmTM FC (Thermo Scientific, Waltham, MA, USA).

Estimation of lipid peroxidation
The stored testis was removed from −80°C (frozen tissues within ice-cold phosphate buffer) was comminuted ultrasonically to make tissues homogenate, then centrifuged at 1000×g for 15 min twice and the supernatant was performed to analyze antioxidant activities.The levels of GSH, MDA, and SOD were analyzed by commercial reagent kits according to the manufacturer's protocols.The expressed as MDA (nmol/mg protein) level at 532 nm.GSH (nmol/mg protein) content was monitored at 412 nm, while SOD (U/mg protein) activity was analyzed at 560 nm.

H&E and TUNEL staining
For evaluation of testis and epididymis tissues, which were fixed in a 10% formalin solution for more than 24 h and then embedded in paraffin, both histological and morphometric analyses were performed on 5-μm sections  cut on a rotary microtome.After staining via H&E dye kits, subsequently performing an observation histopathological changes used light microscope (Leica, DM750, Germany).
To further evaluate the in situ apoptosis in testes, the TUNEL method was performed with apoptosis detection kits (Roche Applied Science, Germany), as previously reported (Xing et al., 2019).Briefly, the testis tissue slices were incubated in 20 mg/mL proteinase K solution for 10 min.Pre-equilibrated slices were subsequently incubated with equilibrium buffer solution and terminal deoxynucleotidyl transferase (TdT).Peroxidase activity in each testis section was stained by diaminobenzidine (DAB).Subsequently, apoptotic cells were photographed using a light microscope (Leica TCS SP8, Germany).

Immunohistochemical (IHC) and Immunofluorescence analysis
Immunohistochemical analysis was executed on testis tissue sections as previously described (Li et al., 2019).In brief, the 5-μm paraffin sections were de-paraffinized and rehydrated and then treated with citrate buffer solutions (0.01 mol/L, pH 6.0).The slices were incubated chamber overnight at 4°C with primary antibodies.Thereafter, slides were counterstained with hematoxylin staining.Immunostaining was monitored digitalized images snapped with an Olympus camera (Tokyo, Japan).
To assess the expression of HO-1 and HSP70 in HSinduced testicular injury, immunofluorescence staining was exerted in testicular sections of HS-induced groups and N group.Briefly, the sections were rehydrated using xylene and different concentration alcohol solutions, subsequently incubated with primary antibodies overnight at 4°C followed by secondary antibody (BOSTER, Wuhan, China).Then, the sections were further stained with 4,6 diamidino-2phenylindole (DAPI) (Vector, Burlingame, CA, USA) for 5 min in the dark.Fluorescence microscope (Leica TCS SP8, Germany) and Image-Pro plus 6.0 software were respectively used to observe and analyze the intensity of immunofluorescence staining.

Western blot analysis
The RIPA lysis buffer was used to extract testicular tissue protein and the BCA protein assay kit (NO.P0010S) was used to determine the concentrations of protein (Beyotime, Jiangsu, China).Western blot analysis was performed as our previous research (Ren et al., 2019).Then membranes were blocked with 5% BSA for 120 min.Subsequently, the membranes were incubated overnight with primary antibodies at 4°C, the level of β-actin (1:1000) was assessed as a loading control, then the secondary antibody (1:2000) was allowed to bind to the primary antibody for 60 min at room temperature.Signals were captured by Emitter Coupled Logic (ECL) substrate (Pierce Chemical Co., Rockford, IL, USA).The intensities of each band were quantified and analyzed by using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis
All data referenced were expressed as the mean ± SD of the indicated number (N) of independent experiments.Differences among experimental groups were performed with one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison post hoc test.Statistical analysis was performed using GraphPad Prism 6.0 software (RRID: SCR_002798, La Jolla California, USA).p < 0.05, p < 0.01 or p < 0.001 were considered significant differences.

G-Rg3 prevented HS-induced changes on testes and epididymis
After administration on the 7th day, the lower body of the mice was placed at 43°C for 18 min to lead to scrotal hyperthermia.The mice were sacrificed 1 week later and immediately measured the weight of the removed testes and epididymis.As showed in Fig. 3A, after HS exposure, the significant shrinkage of testicular and epididymis tissues, and these two organ indices decreased in comparison with the N group (p < 0.001).However, G-Rg3 (5 mg/kg) relieved the reduction of testis and epididymis indices.G-Rg3 at high dosage (10 mg/kg) significantly reversed HS-induced these indices decrease (p < 0.01, p < 0.001).(Figs.3B-3C).

G-Rg3 prevented HS-induced changes on testes and epididymis
After administration on the 7th day, the lower body of the mice was placed at 43°C for 18 min to lead to scrotal hyperthermia.The mice were sacrificed 1 week later and immediately measured the weight of the removed testes and epididymis.As showed in Fig. 3A, after HS exposure, the significant shrinkage of testicular and epididymis tissues, and these two organ indices decreased in comparison with the N group (p < 0.001).However, G-Rg3 (5 mg/kg) relieved the reduction of testis and epididymis indices.G-Rg3 at high dosage (10 mg/kg) significantly reversed HS-induced these indices decrease (p < 0.01, p < 0.001).(Figs.3B-3C).

G-Rg3 prevented HS-induced changes on testes and epididymis
After administration on the 7th day, the lower body of the mice was placed at 43°C for 18 min to lead to scrotal hyperthermia.The mice were sacrificed 1 week later and immediately measured the weight of the removed testes and epididymis.As showed in Fig. 3A, after HS exposure, the significant shrinkage of testicular and epididymis tissues, and these two organ indices decreased in comparison with the N group (p < 0.001).However, G-Rg3 (5 mg/kg) relieved the reduction of testis and epididymis indices.G-Rg3 at high dosage (10 mg/kg) significantly reversed HS-induced these indices decrease (p < 0.01, p < 0.001).(Figs.3B-3C).

G-Rg3 ameliorated HS-induced histopathological alterations
Histologically, testicular tissues from mice in the N group showed quite normal with dynamic spermatogenesis (Fig. 3D).Contrary, a single treatment of HS caused severe damage to spermatogenic cells, with vacuolated nuclei less larges primary spermatocytes and absence of sperm formation, atrophic seminiferous epithelium cells appear to be degeneration, vacuolization, and disorganization.Interestingly, all the testicular structure and seminiferous tubules were almost completely restored to a normal level by G-Rg3 treatment, especially at a high dosage of 10 mg/kg.

G-Rg3 reversed HS-induced testosterone concentration
Testosterone is an important indicator to evaluate male testicular function.Simultaneously, also as a biomarker for spermatogenesis and sperm maturation (Wang et al., 2009).As shown in Fig. 4A, the serum testosterone concentration of the HS group was significantly lower than the N group levels of GSH and SOD in the testis homogenate of the HS group were significantly lower than those of the N group (p < 0.001), while the G-Rg3 treatment at the dose of 5 and 10 mg/kg inhibited the decrease of GSH and SOD levels to varying degrees (p < 0.01).Meanwhile, G-Rg3 significantly inhibited HS-induced MDA elevation (p < 0.05, p < 0.01).
In order to further confirm the generation of oxidative stress in vivo, immunofluorescence (Figs.5A-5D) was employed to confirm the expression of HO-1 and HSP70 in testis tissues.The HS group showed the overexpression of these protein levels (p < 0.01), and treatment with G-Rg3 significantly attenuated at both 5 mg/kg and 10 mg/kg, respectively (p < 0.05, p < 0.01).

G-Rg3 alleviated HS-stimulated apoptosis
In order to verify whether HS-induced spermatogenic damage was associated with apoptosis, TUNEL staining was used to observe the changes in nuclear morphology.As shown in Figs.6A, 6B, clear and large brown color staining of the nucleus was discovered around the sperm cells in the testes of the HS group.Upon exposure of the testis to HS, the nuclear chromatin weakly and seriously affects spermatogenesis.In contrast, the number of TUNEL-positive cells was extremely reduced by G-Rg3 pre-treatment.Preliminary evidence showed that G-Rg3 exerted anti-apoptotic effects in HS-induced damage (p < 0.01).
Additionally, the results of immunohistochemistry also showed that the evidently reduced the expression of Bcl-2 and elevated the expression of Bax by HS-induced spermatogenic cellular apoptosis (Figs.6C-6E).However, these changes could be significantly mitigated by G-Rg3 treatment (p <0.05, p <0.01).
Furthermore, we further examined the expression levels of apoptotic protein in testis tissues by western blot analysis.As shown in Figs.6F-6J, HS exposure increased the protein level of Bax and reduced the expressions of Bcl-2 and Bcl-XL in the testis tissues, respectively (p < 0.01).But the decrease of Bax (p < 0.01) was simultaneously attenuated by supplement with G-Rg3 on the 14th day, while significantly improved the protein Bcl-2 and Bcl-XL expression (p < 0.01).These findings fully demonstrate that G-Rg3 treatment prevented HS-induced testicular tissue apoptosis.

G-Rg3 regulated the MAPK signaling pathways
In the present study, we detected the expression of MAPK family members by western blot analysis and explore the underlying mechanism of G-Rg3 on HS-induced spermatogenesis disorders in mice.As shown in Figs.7A-7C, the expressions of JNK, ERK and p38 MAPK phosphorylation proteins increased (p < 0.01) in the mice of HS group.However, the effect of G-Rg3 was the most significantly at high dosage of 10 mg/kg (p < 0.01).These results indicated that the protective effects of G-Rg3 treatment on HS-induced spermatogenesis disorders is closely related to the MAPK signaling pathway.

Discussion
With the changes in climatic conditions and living environment, a series of diseases caused by HS is gradually gaining importance (Dahl et al., 2019).A growing body of evidence indicates that HS-induced by high-temperature results in deleterious effects such as high blood pressure, cardiac (Chen et al., 2020), gastric mucosa lesions (Qin et al., 2007), and multiorgan dysfunction (Havenith et al., 1995) in human.However, it is worth noting that spermatogenesis is a temperature-dependent process and that scrotal temperature is one of the most important causes of impaired spermatogenesis and male infertility (Liu et al., 2016;Rao et al., 2015).Therefore, it is important to prevent an elevated temperature in testis caused by external environmental factors.In the present study, the protective effects of G-Rg3 against HS-induced testicular damage were investigated in male mice.HS can cause a decrease in the testis and epididymis indices and morphological atrophy and lead to oxidative stress injury and spermatogenic cell apoptosis.Our results showed that G-Rg3 treatment (5 and 10 mg/kg) for consecutive two weeks improved changes in basic indicators and inhibited the expression level of oxidized and apoptosis proteins and were related to the activation of MAPK signaling pathways.More and more evidence showed that dietary medicinal food has beneficial effects on health, especially under HS.P.
ginseng has gained its popularity as an adaptogen for thousand years due to its triterpenoid saponins (call ginsenosides).These ginsenosides are unique and classified, has numerous powerful pharmacological effects, such as on the central nervous system, endocrine, immunomodulatory, and cardiovascular systems (Lee et al., 2019;Qi et al., 2011).PPD-type ginsenosides are the most enriching saponins in Asian ginseng and American ginseng, including ginsenosides Rb1, Rc, Rb2, and Rd, whereas ginsenoside Rg3 is abundantly present in Red or Black ginseng.More importantly, deglycosylation and dehydration reactions of glycosylation of C-3 and C-20 hydroxyl groups by heat processing, acid treatment or biotransformation, can production of rare saponins, such as ginsenoside Rg3, Rk1, Rg5, F2, and Compound K, etc.These transformed ginsenosides further contribute to the chemical and pharmacological activity diversity of ginsenosides.In this study, a rare saponin G-Rg3 was prepared by using PPD-type ginsenoside Rd as a reaction substrate and used to investigate the protective effect of scrotal HS-induced on spermatogenic damage.Based on previous studies found that G-Rg3 has multiple Bcl-XL).Tissues were homogenized and processed into western blot analysis of these protein expressions, and β-actin protein was used as loading control; (G-J) Quantification of relative protein expression was performed by densitometric analysis (N = 6 each group).Data are expressed as mean ± SD. **p < 0.01 vs. N group; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. HS group.
beneficial effects in acute experimental animal models (Zhou et al., 2018).
Recent studies on various animal tissues and experimental models have indicated that HS can severely and adversely affect the isolation of spermatogenic cells by disrupting spermatogenesis and altering oxidative metabolism (Ghasemi et al., 2009).Prior to the present study, Kopalli et al. (Kopalli et al., 2019) have confirmed that Korean red ginseng extract (enriched ginsenoside Rg3) protects the scrotal from chronic intermittent HS-induced spermatogenic damage and help to recover testicular function by restoring intrinsic anti-oxidant defenses.Similarly, in the current histopathological examination of tissues, the slides stained with H&E revealed disordered seminiferous tubules and spermatocytes and spermatids severe lesions or even disappeared in the HS group.However, supplement with G-Rg3 restored HS-induced tissue damage with varying degrees, especially the 10 mg/kg of G-Rg3 almost full recovery.
Meantime, HS-induced oxidative stress is mainly caused by lipid peroxidation of cell membrane potential, intracellular ROS, and MDA concentrations in the testes, which can be closely related to the degree of membrane system damage (Li et al., 2014).Recent research has also illustrated that hypoxia stress, such as HS, causes a rapid increase of ROS, and is correlated with the upregulation of the SOD and GSH (Kim et al., 2013a).More importantly, antioxidant enzymes also limit the toxicity associated with free radicals or ROS, which disturb the balance of redox reaction in vivo (Li et al., 2018).In the current work, the MDA level expression in the HS group was significantly increased with compared to the N group, and our experimental data showed that G-Rg3 treatment (5 and 10 mg/kg) for consecutive two weeks reversed elevation of MDA, as well as reduction of GSH and SOD in the testis.
Apart from these biochemical parameters, it is worth noting that HO-1 and HSP-70 are the most important cytokines for evaluating peroxidative damage (Farag et al., 2019).Evidence also suggests that HIF-1α is currently the single most important indicator of impaired testicular function, which is a central regulator responsible for the regulation of pathological inducible response (Li et al., 2017).In agreement, our present study showed a significant reduction in the expression of antioxidative proteins related to spermatogenesis such as HO-1, HSP-70, and HIF-1α levels in HS groups, and treatment with G-Rg3 significantly ameliorated these changes by immunofluorescence staining and western blot analysis.Collectively, these results provide more strong evidence that G-Rg3 may in preventing HSinduced oxidative damage in the mice testis.
Importantly, there are few studies on the specific mechanism of HS-induced spermatogenic damage.MAPKs is known to play a critical role in the signal cellular programs of transduction, and also throughout evolution in many physiological processes (M and Pp, 2011), JNK, ERK, and p38 are important proteins of the MAPK family, engages in cytokine synthesis and differentiation, especially closely related to the production of ROS and oxidative stress (Azadeh et al., 2020).Activated MAPK also can regulate cell growth, motility, and mitochondrial pathway-mediated apoptosis (Sui et al., 2014).Thus, we speculate that the MAPK signaling pathway may be involved in regulating oxidative stress and apoptosis induced by HS.In current experiments, HS exposure increased the protein expression levels of JNK, ERK, and p38 in testis tissues.However, G-Rg3 treatment significantly reduced the expression of these proteins.The present study confirmed the negative regulation of G-Rg3 on the MAPK signaling pathway (Fig. 8).
Mounting evidence suggests that MAPK can alter various oxidative enzymes and involved mitochondrial-induced apoptosis, and the expression of Bax and Bcl-2 apoptosis proteins is also closely related to JNK phosphorylation (Ki et al., 2013).Meantime, previous studies have confirmed that experimental cryptorchidism can also cause apoptosis (Shikone et al., 1994).As showed in this experiment, our data demonstrated that G-Rg3 treatment ameliorates the expressions of effector Bax, Bcl-2, and Bcl-XL, showing that G-Rg3 has a great anti-apoptotic activity to relieve HSgerm cell apoptosis.Furthermore, cell apoptosis in vivo is quite complicated and can be regulated by interacting with one another through various mechanisms (Sinha Hikim et al., 2003).In the current study, TUNEL assay and immunohistochemical staining also verified the anti-apoptotic effect of G-Rg3.However, it is worth noting that organ damages induced by HS, may be regulated by complex signal pathways and various other factors in vivo.The protective effects of G-Rg3 on the male testis under HS conditions need further investigations both in vivo and in vitro.
In conclusion, this research used the efficient thermal deglycosylation of ginsenoside to prepared G-Rg3 and further demonstrated that the protective effects and underlying mechanism of G-Rg3 against HS-induced spermatogenic damage in mice by suppression of ROSmediated oxidative stress and apoptosis via partly regulation of the MAPK signaling pathway.G-Rg3 can be an important natural ingredient, which has potential therapeutic for the clinical treatment of male infertility or infertility caused by hyperthermia.

FIGURE 6 .
FIGURE 6. G-Rg3 regulated on apoptosis in HS mice and inhibited the expression of apoptosis-related protein induced by HS. (A) TUNEL assay of apoptotic spermatogenic cell; (B) Quantitative analysis of TUNEL-positive cell; (C) Testis tissues (5-μm section) from each group were processed for Bax and Bcl-2 immunohistochemical staining as described in materials and methods; Representative staining images were shown (magnification ×400); (D-E) Quantitative analysis of Bax and Bcl-2 positive cells; (F) Western blot analysis of apoptosis-related (Bax, Bcl-2 and Bcl-XL).Tissues were homogenized and processed into western blot analysis of these protein expressions, and β-actin protein was used as loading control; (G-J) Quantification of relative protein expression was performed by densitometric analysis (N = 6 each group).Data are expressed as mean ± SD. **p < 0.01 vs. N group; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. HS group.

FIGURE 7 .
FIGURE 7. Administration with G-Rg3 regulated MAPK signaling pathway induced by HS. (A) Western blot analysis of MAPKs.Tissues were homogenized and processed into western blot analysis of ERK, JNK, p38 MAPK, and their phosphorylated forms, and β-actin as loading control; (B-D) Quantification of relative protein expression was performed by densitometric analysis (N = 6 each group).Data are expressed as mean ± SD. **p < 0.01 vs. N group; # p < 0.05, ## p < 0.01 vs. HS group.