Loganin alleviates testicular damage and germ cell apoptosis induced by AGEs upon diabetes mellitus by suppressing the RAGE/p38MAPK/NF‐κB pathway

Abstract Diabetes mellitus (DM) damages male reproduction at multiple levels, such as endocrine secretion, spermatogenesis and penile erection. We herein investigated the protective effects and mechanism of loganin targeting the advanced glycation end products (AGEs)/receptor for AGEs (RAGE)/p38 mitogen‐activated protein kinase (p38MAPK)/NF‐κB signalling pathway. Loganin relieved the general DM symptoms and decreased the blood glucose level of KK‐Ay DM mice. Haematoxylin‐eosin staining demonstrated that loganin ameliorated testicular histology and function and enhanced the activities of testis‐specific markers lactate dehydrogenase (LDH), acid phosphatase (ACP) and gamma‐glutamyl transferase (γ‐GT). Loganin also showed evident anti‐oxidative stress, anti‐apoptotic and anti‐inflammatory effects on DM‐induced reproductive damage by restoring glutathione (GSH) level and superoxide dismutase (SOD) activity, as well as reducing reactive oxygen species (ROS) level and Bax/Bcl‐2 ratio in vivo and in vitro. Western blotting exhibited that loganin significantly inhibited the AGEs/RAGE/p38MAPK/NF‐κB signalling pathway. Acridine orange and ethidium bromide staining (AOEB) and Western blotting showed that loganin in combination with inhibitors of RAGE, p38MAPK and NF‐κB exerted stronger anti‐apoptotic effects on AGE‐induced GC‐2 cell damage compared with loganin alone. In conclusion, loganin can protect against DM‐induced reproductive damage, probably by suppressing the AGEs/RAGE/p38MAPK/NF‐κB pathway.


| INTRODUC TI ON
Diabetes mellitus (DM) is a prominent metabolic disorder affecting 425 million people worldwide in 2017, a number expected to rise to 629 million by 2045. 1 Although the effects of DM on the reproductive system have long been controversial, it is now accepted that long-term uncontrolled DM with sustained high blood glucose level can cause testicular damage by inducing various micromolecular changes such as erectile dysfunction, retrograde ejaculation, loss of libido and abnormal sperm production that may lead to male infertility. [2][3][4] A retrospective study reported subfertility in 51% of male diabetics. 5 Another study showed that diabetic males had significantly higher rates of primary (16%) and secondary (19.1%) infertility than those of subjects without DM. 6 Although DM is an important cause for male infertility, the underlying mechanism is still unclear. The apoptosis of germ cells and oxidative stress are two major events involved in DM-induced testicular damage. In DM rats, lipid peroxidation and reactive oxygen species (ROS) overproduction are promoted, and the activities of superoxide dismutase (SOD), catalase and glutathione (GSH) are inhibited. 7,8 Besides, Chen et al reported that the apoptotic rate of germ cells and Bax/Bcl-2 ratio in DM rats exceeded those of normal controls. 9 Advanced glycation end products (AGEs) produced by non-enzymatic reactions between the sugar and amino groups of proteins under hyperglycaemic conditions predominantly trigger oxidative stress and cell dysfunction upon numerous diabetic complications.
Mallidis et al also identified these compounds and receptor for AGEs (RAGE) in the male reproductive system, including testes, epididymis and sperm. 10,11 Karimi et al further confirmed that AGEs played a key role in increasing oxidative stress in the case of reproductive system dysfunction. 12,13 P38 mitogen-activated protein kinase (MAPK) is a part of an essential intracellular signalling pathway involved in cell growth, differentiation, development and apoptosis. The phosphorylation of p38 MAPK induced by AGE-RAGE binding has also been shown to play a central role in various forms of DM-induced reproductive dysfunction, including testicular damage and erectile dysfunction. 7,9,14 According to the theory of traditional Chinese medicine (TCM), kidney Yin deficiency is the core pathogenesis of DM-induced reproductive injury, and consequently, reproductive disorders are commonly treated by tonifying the kidney. 15 Cornus officinalis Sieb. et Zucc. (family: Cornaceae) has often been used in TCM formulations such as Liu-Wei-Di-Huang pills and its derivatives, which can nourish the liver and kidneys, treat impotence, remove internal heat, etc. 16 Cornus officinalis (CO) extract, iridoid glycosides and single compound have been reported to alleviate the damage to diabetic target organs such as the kidneys and testes. 9,17,18 As a primary bioactive monomer extracted from CO iridoid glycoside, 16 loganin can inhibit inflammation 19,20 and protect against DM-induced nephropathy 21 and neuropathy. 22 However, its influence on testicular damage in the context of DM has seldom been referred hitherto. Therefore, we herein, for the first time, used spontaneous type 2 DM (T2DM) model KK-Ay mice and GC-2 cells to explore the function and mechanism of loganin in relieving DM-induced testicular damage and sperm cell apoptosis targeting the AGEs/RAGE/p38MAPK/NF-κB signalling pathway. The results would provide novel insights into the potential use of loganin to prevent male infertility upon T2DM.

| Reagents and antibodies
Loganin ( Figure 1A; 98%  To prepare AGEs, 50 mg/mL bovine serum albumin (BSA) was incubated with 0.5 mol/L D-glucose at 37°C for 12 weeks in a sterile environment without light. After the formation of AGEs, the solution was dialysed in 10 mmol/L pH 7.4 phosphate-buffered saline (PBS) to remove unreacted glucose, and AGE content was determined using an ELISA kit.

| Animal model establishment
Fourteen-week-old male KK-Ay mice and C57BL/6J mice (Licence Number: 2014-0004) were purchased from Beijing Hua Fukang Biotechnology Co., Ltd. The flow chart of animal study design is shown in Figure 1B. KK-Ay mice were fed high-fat diet (458 kcal/100 g, containing 10% fat), and C57BL/6J mice were fed normal diet. All mice were housed at (25 ± 1)°C and humidity of (55 ± 5)% in a regular 12 h/12 h light/dark cycle to acclimate for two weeks prior to experiments. The animal studies were conducted in accordance with protocols approved by the Animal Ethic Committee of Nanjing University of Chinese Medicine (code: ACU-13(20161011)). KK-Ay mice were randomly divided into four groups (n = 6): DM group, aminoguanidine group (100 mg/kg/d) as the positive control, low-dose loganin group (50 mg/kg/d) and high-dose loganin group (100 mg/kg/d). All these groups were fed F I G U R E 1 Loganin ameliorated general diabetic symptoms in DM mice. A, Structure of loganin. B, Flow chart of animal study design. C, 24-h water intake in the fourth and eighth weeks of experiment. D, 24-h urine volumes in the fourth and eighth weeks. E, 24-h food consumptions in the fourth and eighth weeks. F, Bodyweights in the fourth and eighth weeks. G, Fasting blood glucose levels in the fourth and eighth weeks. H, Serum GSP level at the end of experiment. Significance: ## P < .01 vs normal group; *P < .05, **P < .01 vs model group high-fat diet. Each treatment group was orally given corresponding agent for eight weeks. Six C57BL/6J mice that were fed normal diet and administered with an equal volume of saline were set as a control group. Bodyweight, 24 hours food consumption, 24 hours water intake, 24 hours urine volume, serum insulin level and fasting blood glucose level were measured every four weeks. At the end of experiment, all mice were killed by cervical dislocation, and blood samples were drawn from the orbit. The blood samples were left still at room temperature for 20 minutes and centrifuged at 3000 g for 15 minutes, and then the serum was collected. The testes of each animal were collected and weighed.

| Determination of live sperm rate
The live sperm rate was determined according to the method described by Giribabu et al. 23 The epididymides were homogenized and suspended with PBS. Then, 100 μL of the suspension was mixed with an equal volume of 1% trypan blue in the same medium. Live sperm totally excluded the dye, whereas dead sperm accumulated the dye and exhibited blue heads. Live sperm were analysed under a light microscope with 200× magnification and expressed as a percentage of the total sperm count.

| Histological analysis and transmission electron microscopy (TEM)
The testes and kidneys were fixed in 10% formalin solution and then embedded in paraffin. The paraffin blocks were cut into 5-μm-thick sections and stained with haematoxylin and eosin (HE). Photographs were taken in a blinded manner from randomly selected fields, and representative images of the sections are shown. Testicular pathology was analysed by pathologists according to the hierarchical arrangement and structure of spermatogenic cells, interstitial cell hyperplasia, interstitial vasodilation, hyperaemia, haemorrhage and inflammatory cell infiltration for semi-quantitative scoring. Kidney pathology was scored on the basis of renal structural integrity, glomerular mesangial hyperplasia, inflammatory cell infiltration and renal tubular epithelial lesions. A score of 0 indicates no lesions, and a score of 6 was assigned to the most serious lesions. For kidney TEM, the renal cortex was cut into 1 mm × 1 mm chunks and fixed with 2.5% glutaraldehyde solution at 4°C for 24 hours, followed by gradient dehydration and imaging of the glomerulus in random fields and representative podocytes.

| Measurement of AGEs in testes
AGE levels in the testes were measured using ELISA kit (Nanjing Yifeixue Biotechnology Co., Ltd.) according to the manufacturer's protocol. Briefly, 96-well plates were coated with primary antibody and 50 μL of the standard, and control or sample was added into each well of the plate and incubated for 30 min at 37°C. After washing three times, 100 μL of enzyme-conjugated solution was added and incubated for 30 minutes at 37°C. After another three washes, p-nitrophenyl phosphate substrate diluted in glycine buffer was added.
The absorbance was read at 450 nm using a microplate reader.

| Measurement of oxidative stress and testicular markers
The levels of ROS, GSH and the activities of SOD, lactate dehydrogenase (LDH), acid phosphatase (ACP) and gamma-glutamyl transferase (γ-GT) in testes and GC-2 cells were measured using kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Intracellular ROS production was estimated by using 2,7-dichloro- Phenol and 4-aminoantipyrine can be oxidized by potassium cyanide into red quinone derivatives in alkaline solution. The derivatives were quantified using a microplate reader at 520 nm. The experiments were performed in triplicate.

| Evaluate of apoptotic testicular cells in KK-Ay mice by TUNEL staining
Apoptotic testicular cells were measured using TUNEL assay with a kit purchased from Beyotime Biotechnology Co. Ltd. (batch No. C1086, China). Testicular tissue was fixed in 10% paraformaldehyde, embedded in paraffin and then sectioned into 5 μm thick. Briefly, the sections were deparaffinized and rehydrated in xylene and ethanol, followed by incubation with proteinase K working solution at 37°C for 20 minutes. After rinsing with PBS, the samples were reacted with TUNEL reaction mixture (50 µL) at 37°C for 1 hour and rinsed with PBS to stop the reaction. TUNEL-positive cells were considered apoptotic and quantitatively analysed with ImageJ software.

| Evaluate of GC-2 cell apoptosis by acridine orange and ethidium bromide (AO/EB) staining and flow cytometry (FCM)
AO/EB dual staining was performed at a 1:1 ratio to evaluate the morphological changes of cells due to apoptosis, and flow cytom-

| Statistical analysis
Data were collected from repeated experiments and are represented as mean ± standard deviation (SD). One-way ANOVA was used to determine whether a difference was present and if so, the post hoc Tukey's test was used to analyse the differences between groups using SPSS 19.0 software. P < .05 was considered statistically significant. week, which were significantly reduced by loganin ( Figure 1G,H).

| Loganin mitigated kidney and testicular lesions of KK-Ay mice
Since reproduction is closely related to renal function in TCM, we also evaluated the effects of loganin on kidney lesions in KK-Ay DM mice. HE staining and TEM were used to assess morphological changes, which showed obvious lesions with thickening of the glomerular basement membrane, glomerular hypertrophy and fusion of podocyte foot processes in model mice (Figure 2A,B).
In the testis, the control mice exhibited normal testicular structure with intact seminiferous tubules and organized germ cells in concentric layers. Spermatogenic cells at various stages of division (from spermatogonia to spermatid) were observed in neatly arranged seminiferous tubules, with small amounts of interstitial cells in the lumen ( Figure 3A). In the model group, the seminiferous tubules were disrupted, accompanied by germ cell degeneration, structural shrinkage and separation from each other ( Figure 3B). However, these pathological changes were alleviated to different extents by treatment with positive drug aminoguanidine or loganin for eight weeks (Figures 2A,B and 3C-E). The testicular lesion scores are shown in Figure 3F.
Furthermore, the expressions of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in the renal cortex were measured to evaluate apoptosis, and WT1 expression was measured to assess podocyte loss. The Bax/Bcl-2 ratio of the model group significantly exceeded that of the normal group, which was reversed by using aminoguanidine or loganin ( Figure 2C). WT1, a specific marker protein for podocytes, was down-regulated in the model group in comparison with that of the normal group, which was restored by aminoguanidine or high-dose loganin treatment ( Figure 2D).

| Loganin attenuated apoptosis in testes of KK-Ay mice and AGE-induced GC-2 cells
The TUNEL assay revealed numerous apoptotic cells in the testes of the model group, and the apoptosis was mitigated by aminoguanidine F I G U R E 2 Loganin showed renal protective effects in KK-Ay mice. A, HE staining of kidney tissues. B, Transmission electron microscopy of glomeruli. C, Western blot analyses of Bax and Bcl-2 protein expression in kidney cortex. The Bax/Bcl-2 ratio was calculated and analysed. D, Western blot analyses of WT1 protein expression in kidney cortex. Bars represent the mean ± SD, n = 3. Significance: ## P < .01 vs the normal group, *P < .05, **P < .01 vs the model group or loganin administration ( Figure 4A,B). Additionally, the Bax/Bcl-2 ratio of model mice was significantly (P < .01) elevated ( Figure 4C) compared with that of the normal control group, which was effectively reversed by aminoguanidine or loganin administration (P < .01). Therefore, loganin exerted an anti-apoptotic effect on DMinduced testicular damage. AO/EB staining and FCM were performed to evaluate the in vitro protective effects of loganin on GC-2 cell apoptosis induced with AGEs (200 mg/L) for 48 hours. The apoptosis rate of GC-2 cells after AGEs treatment was significantly higher than that of the control group. However, pre-treatment with loganin (0.1, 1 and 10 μmol/L) decreased the apoptosis rate significantly (P < .01) in comparison with that of the AGEs group ( Figure 5A-D). Furthermore, the Bax/ Bcl-2 ratio was increased 9.7-fold after AGE stimulation (P < .01), which was effectively reversed by loganin administration (P < .01, Figure 5E,F). Thus, loganin exerted an anti-apoptotic effect on GC-2 cells stimulated with AGEs.

| Loganin improved oxidative stress and inflammatory responses and down-regulated testisspecific enzymes
Testis-specific marker enzymes LDH, ACP and γ-GT significantly de-

| Loganin inhibited the activated AGEs-RAGE/ p38 MAPK/p65 NF-κB pathway in DM mice and GC-2 cells
To further explore the mechanism underlying the protective effects of loganin, we measured the levels of AGEs and RAGE in the testes. The phosphorylation of p38 MAPK and p65 NF-κB, one of the downstream pathways for RAGE closely related to the induction of inflammation and apoptosis, was also tested. The model group had higher levels of AGEs in the testes than those of normal controls, but aminoguanidine or loganin attenuated the deposition of AGEs to various degrees ( Figure 7A). Western blot exhibited that compared with normal mice, RAGE expression was elevated in model mice (P < .01), but significantly down-regulated by aminoguanidine or loganin ( Figure 7B). Additionally, the phosphorylation of p38 MAPK and p65 NF-κB was enhanced in model mice compared to that of the normal group, which was significantly attenuated by treatment with aminoguanidine or loganin for eight weeks ( Figure 7C,D).
To further explore the mechanism for the protective effects of NF-κB and nuclear/cytoplasmic ratio (P < .01; Figure 7G). The cells exposed to pre-treatment with loganin (0.1, 1 or 10 μmol/L), especially that at 10 μmol/L, had significantly decreased RAGE expression and phosphorylation of p38 MAPK compared with those of the AGE group ( Figure 7E,F). Additionally, the nuclear translocation of p65 NF-κB was also significantly attenuated in the 10 μmol/L loganin group (P < .01, Figure 7G). with that of the group administered with loganin only ( Figure 7J).
Collectively, the anti-apoptotic effect of loganin was associated with suppression of the AGEs-RAGE/p38 MAPK/p65 NF-κB signalling pathway.

| D ISCUSS I ON
Diabetes mellitus is a chronic metabolic disease causing multiple organ and system dysfunction, including kidney failure, retinopathy and so on. With increasingly younger age of onset, the effect of DM on the male reproductive system has gradually aroused concern. DM may compromise male reproduction function at multiple levels, such as endocrine secretion, spermatogenesis, penile erection and ejaculation. 5 The effects of DM on sperm count, motility and morphology have been widely studied, 24,25 but the results are inconsistent and the treatment or mechanism of DM-induced reproductive damage remains elusive.
In this study, we selected spontaneous DM model KK-Ay mice which had marked obesity, glucose intolerance, insulin resistance and hypertension, all being the characteristics of T2DM phenotype. 26 Zhou et al also reported serious kidney injury in KK-Ay mice, manifested as albuminuria, glomerular hypertrophy and mesangial matrix accumulation. 27 Our study showed that 14-week-old male In summary, loganin significantly mitigated DM-induced reproductive damage by improving diabetic metabolic parameters, protecting testicular structure and function, as well as attenuating oxidative stress, inflammation and apoptosis in the testes. Loganin also reduced the level of AGEs, and inhibited the protein expression of RAGE and its downstream p38MAPK/NF-κB signalling pathway, which may be related to DM-induced oxidative stress, inflammation and apoptosis in the testes. Further in-depth studies are still needed to clarify the mechanism by which loganin protects against DMinduced reproductive disorders.

ACK N OWLED G EM ENTS
This work was financially supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors all declare that they have no competing interests.