Morinda officinalis oligosaccharides alleviated mice chronic unpredictable mild stress induced sexual dysfunction

Abstract

Background

Chronic stress can not only lead to depression-like behavior but also sexual dysfunction. Morinda officinalis oligosaccharides (MOO) is a formula of traditional Chinese medicine commonly used in invigorating the kidney and strengthening Yang, and relieving depression.

Aim

This study was designed to explore the effects and mechanisms of MOO in treating chronic stress-induced depression as well as sexual dysfunction.

Methods

The sucrose preference test, forced swimming test (FST) and novelty-suppressed feeding test (NSFT) were carried out to evaluate the depression status. Sexual behavior was tested on all mice, then the extent of damage to the testicles and epididymis was assessed by H&E staining; Serum sex hormone and neurotransmitters were assessed in the plasma by Enzyme-Linked Immunosorbent Assay. The testicular tissues were applied with the kit for the detection of antioxidant-related indexes and reproductive-related hormones.

Outcomes

The study evaluates the effects of MOO on depression—like behaviors and sexual function levels in CUMS—induced mice by analyzing the behavioral tests, histopathological staining of testis and epididymis, sex hormones, antioxidant capacity, neurotransmitter levels, and sexual behavior abilities of mice in each group.

Results

CUMS led to mice depression and plasma neurotransmitter levels decreased. Accompanying sexual dysfunction in depressed mice was also manifested in many aspects. Compared with the control group, the capture latency and mount latency of male mice in model group were significantly prolonged. HE showed that testicular and epididymal tissues of mice in the CUMS group were severely vacuolated. Testicular marker enzymes, antioxidant indexes and sex hormones were disorganized. The sperm concentration and viability in the epididymis of the mice in model group were significantly reduced. It was suggested that MOO could improve the damage caused by CUMS, and improve the sperm quality of the model mice.

Clinical Translation

MOO are promising to be translated into a potential therapeutic drug for clinically improving chronic stress-related depression and sexual dysfunction.

Strengths and Limitations

Multi-dimensional verification confirms that MOO can effectively alleviate depressive states and sexual dysfunction in CUMS-induced mice. Future studies should explore the in-depth mechanisms underlying its antidepressant and anti-sexual dysfunction effects based on relevant signaling pathways.

Conclusion

These results suggest that MOO can regulate sexual dysfunction and play a protective role in neurodevelopment during CUMS by regulating sex hormones.

Introduction

Morinda officinalis, one of the 4 traditional southern medicines in China, comes from the plant Morinda officinalis dry root.^1,2 The main treatment of impotence and spermatorrhea, uterine cold infertility, menstrual disorders, abdominal cold pain, rheumatism paralysis and impotence and weakness of muscles and bones.^3,4 Morinda officinalis contains a variety of chemical constituents, mainly including sugar, cyclic allyl ether terpenes, amino acids, anthraquinones and volatile components.⁵ It is found that the sugar components in Morinda officinalis can not only improve immunity, enhance memory, antioxidant antiaging effect and show good antidepressant activity.⁶ The researchers of Morinda officinalis in the oligosaccharide composition of a series of studies, that Morinda officinalis in the oligosaccharide composition for its antidepressant main material basis. Morinda officinalis oligosaccharide (MOO) Capsules, which are mainly composed of MOO, have been approved to be marketed as the fifth kind of anti-depression new drugs in Chinese traditional medicine with independent intellectual property rights, but also opened up the traditional Chinese medicine in the field of antidepressant modernization process.

Depression is a common mental disorder characterized by significant and persistent depressed mood, high prevalence, high relapse rate and high disability rate, and is a global public health problem.⁷ The World Health Organization (WHO) officially reports that the lifetime prevalence of depression is 10%-15%, affecting more than 120 million people globally, and the number of suicides due to depression is about 800 000 per year.⁸ According to WHO’s estimation, as of 2030, depression will become the primary burden of disease globally.⁹ The etiology of depression is complex and is related to a variety of biochemical, genetic, social, psychological, and cultural factors.¹⁰ Current clinical drugs for depression often cause patients to experience adverse reactions such as headache, diarrhea, weight gain, loss of appetite, sexual dysfunction.¹¹ Therefore, drugs for the treatment of depression must give due consideration to their long-term efficacy and possible side effects.

Due to long-term anxiety, depression and the adverse effects of antidepressant treatment, the sexual function of male patients with depression is often affected, resulting in reduced libido, premature ejaculation and other sexual function problems, and leading to erectile dysfunction (ED).¹² Erectile dysfunction can aggravate the patient’s anxiety and depression, further aggravating the depression condition, and the 2 cause and effect each other, forming a vicious circle. At present, the commonly used clinical anxiolytic and depressant drugs have more adverse effects and higher addiction and dependence, which are not suitable for long-term application, and the oral drugs used for ED also have poor long-term efficacy and so on. Therefore, there is an urgent need to develop new natural antidepressants with new targets and fewer adverse effects. Chinese medicine has rich experience in the treatment of depression. Chinese medicine categorizes depression and ED as “impotence with depression,” with liver qi stagnation as the main pathology, and the clinical method of liver detoxification is often used to improve the patient’s symptoms.¹³ Many experimental studies have shown that traditional Chinese medicine has a certain therapeutic effect on depression and fewer side effects. Therefore, in this study, on the basis of the antidepressant effect of MOO we studied the improvement of sexual function in mice, and evaluated its effect on the improvement of depressive symptoms and sexual function in depressed ED mice, with a view to providing a new idea for the clinical prevention and treatment of depression and ED.

Methods

Drug administration

The drugs used to prepare the MOO decoctions were purchased from Beijing Tong Ren Tang Group. The treatment dosage for mice was 9.1 times that for patients.¹⁴ For instance, a patient needs 0.6 g herbs per 70 kg body weight every day. Then, each mouse needs 25 mg/kg herbs, which is the low dose of MOO (MOO-L). The medium dose (MOO-M) is twice the low dose. The high dose (MOO-H) is twice the medium dose. In the same way, the daily dose of fluoxetine required for an adult (20 mg/70 kg) was converted into the dose of fluoxetine for mice (0.1786 mg/100 g). Fluoxetine is diluted with 0.9% saline. Institute of Cancer Research (ICR) mice were gavaged with the prepared MOO-L (25 mg/kg), MOO-M (50 mg/kg), MOO-H (100 mg/kg), Flu (12 mg/kg) or 0.9% saline forx 28 days.

Animals and treatments

ICR mice (8 weeks old, 28-32 g) were purchased from Beijing Beiyou Biology. All the mice were housed under a controlled environment with a room temperature at 22 °C, a 12 h light/dark cycle, and with unlimited access to water and food. All animal experiments were carried out in accordance with the rules for the Laboratory Animal Ethics Committee of Beijing Union University.

One week after the acclimation of the environment, the mice were randomly divided into the control group, MOO-L, MOO-M, MOO-H, Flu, CUMS model group (n = 12). For the group, 6 mice were kept in each cage. CUMS was applied to the model group mice for 4 weeks. Two different physical stimulations were applied to the CUMS model mice daily for 4 weeks, as summarized in Table 1.¹⁵ After 4-week treatment, SPT, FST and NSFT were arranged to carry out.

Table 1.

Daily stimulations summary from Day 1 to Day 28.

Day	Stimulations	Day	Stimulations
1	Fasting, 12 h + Clamps hold the tail 6 min	15	Restraint stress by restraint cages, 4 h + Fasting, 12 h
2	All night lighting + Swimming in hot water, 45 °C 5 min	16	All night lighting+ Clamps hold the tail 6 min
3	Strange Odor Pepper, 12 h + Restraint stress by restraint cages, 4 h	17	Swimming in hot water, 45 °C 5 min + Restraint stress by restraint cages, 4 h
4	Damp bedding, 12 h + Swimming in hot water, 45 °C 5 min	18	Fasting, 12 h + Damp bedding, 12 h
5	Clamps hold the tail 6 min + All night lighting	19	Strange Odor Pepper, 12 h + Clamps hold the tail 6 min
6	Fasting water + Restraint stress by restraint cages, 4 h	20	All night lighting + Restraint stress by restraint cages, 4 h
7	Strange odor Pepper, 12 h + Clamps hold the tail 6 min	21	Fasting water + Swimming in hot water, 45 °C 5 min
8	Restraint stress by restraint cages, 4 h + All night lighting	22	Damp bedding, 12 h + Strange Odor Pepper, 12 h
9	Fasting+ Swimming in hot water, 45 °C 5 min	23	Restraint stress by restraint cages, 4 h + Fasting, 12 h
10	Strange odor Pepper, 12 h + Restraint stress by restraint cages, 4 h	24	Clamps hold the tail 6 min + Swimming in hot water, 45 °C 5 min
11	Damp bedding, 12 h + Clamps hold the tail 6 min	25	Damp bedding, 12 h + Fasting, 12 h
12	Swimming in hot water, 45 °C 5 min + All night lighting	26	Restraint stress by restraint cages, 4 h + Swimming in hot water, 45 °C 5 min
13	Clamps hold the tail 6 min + Fasting, 12 h	27	Clamps hold the tail 6 min + Damp bedding, 12 h
14	Swimming in hot water, 45 °C + Damp bedding, 12 h	28	Strange odor Pepper, 12 h + Swimming in hot water, 45 °C

Behavioral tests

The SPT was used to assess the degree of pleasure loss in mice. Mice were subjected to sucrose water acclimatization prior to the start of the experiment. Day 1, in each cage placed 2 water bottles containing sucrose water at a concentration of 1. Day 2, 2 bottles were filled with 1% sucrose water and distilled water. Day 3, the mice were first fasted and dehydrated for 24 h. Day 4, 2 bottles containing 1% sucrose water and distilled water, weigh-in and recorded, were placed in each cage. The placement of the 2 bottles was changed after 6 h to prevent the possible effects of preference for a side on drinking behaviors. After 12 h, the 2 bottles were removed and weighed to calculate the consumption of sucrose water and distilled water. Sucrose water preference (%) = sucrose water consumption/(sucrose water consumption + water consumption) × 100%.¹⁶

The FST are conducted by placing mice in a confined environment where they experience intense struggle and despair without the ability to escape. Over time, the mice exhibit a characteristic resting state, which is considered a state of behavioral despair. The day before the experiment, the mice are placed in the water for a 15-min pre-swim to acclimatize to the environment. At the end of the pre-swimming period, the mice were removed, dried, and given medication or other therapeutic interventions. After a 24-h stress period, mice were placed in the water again for a 5-min test swim. Swimming and resting behaviors of the mice were recorded and analyzed for behavioral changes.¹⁷

The NSFT based on the phenomenon that animals’ food exploration behavior is inhibited in novel environments. Animals show higher alertness and anxious behavior in novel environments, which reduces feeding behavior. The day before the experiment, the animals are placed individually in new cages and subjected to food deprivation, but are ensured to have adequate access to water. The duration of food deprivation is usually 18-24 h. The fasted animal is placed in the new environment and a timer is started to record the time taken from the time the animal enters the new environment to the time it starts eating the novel food.¹⁸

Sexual behavior

Preparation of female mice for mating experiment: Each female rat was injected subcutaneously with 1 ml of estradiol benzoate at 2 mg/mL 48 h before the test, and 1 mL of progesterone at 10 mg/mL 4 h before the test to promote estrus in female rats. After 4 h of the last drug administration, male mice were placed in a glass box 45 * 60 * 60 cm for 5 min to get used to the environment and then the female mice were added. Following sexual behavior was monitored, including the capture latency, capture frequency, mount latency, mount rate and ejaculation frequency (within 30 min).¹⁹

H&E staining

HE staining clearly shows the cellular and histological structure of the tissue. Testicular and epididymal tissues are formalin-fixed, dehydrated, hyalinized, stained with hematoxylin, differentiated, dehydrated and hyalinized, and finally blocked. The lesions were examined under a light microscope at 100× magnification of the testicular and epididymal tissue.

Biochemical assays

Epididymal levels of Lactate Dehydrogenase (LDH), Alkaline Phosphatase (ALP), Acid Phosphatase (ACP), Malondialdehyde (MDA), Superoxide Dismutase (SOD), Glutathione (GSH), and Glutathione Peroxidase (Glutathione Peroxidase) were measured by LDH, ALP, ACP, MDA, SOD, GSH and GSH-PX assay kit (A020-2-2, A059-2-2, A060-2-2, A003-1, A001-3, A006-1-1, A005, Nanjing Jiancheng Bioengineering Institute) using an automatic biochemical detector (BS-240VET, Shenzhen Mindray Bio-Medical Electronics).

Enzyme-linked immunosorbent assay

Hippocampal levels of 5-Hydroxytryptamine (5-HT), Dopamine (DA), Norepinephrine (NE) and Acetylcholinesterase (AChE) were measured by5-HT, DA, NA, ACHE assay kit (CSB-E08365m, CSB-E08661m, CSB-E07870m, CSB-E17521m, CSB-E17521m Wuhan CUSABIO).

Serum levels of Testosterone (T), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) were measured by T, FSH, LH assay kit (E-OSEL-M0003, E-EL-M0511, E-EL-M3053) Wuhan Elabscience).

Sperm quality

Sperm deformities: the tail of the left epididymis was placed in 1 mL of balanced salt solution that had been preheated at 37 °C, clipped and blown to free the spermatozoa, 10 μL of milky-white liquid was added to 990 μL of balanced salt solution that had been preheated at 37 °C, 10 μL of spermatozoa was aspirated on the slide by micropipette gun, and the samples were made into smears in the form of drop pulls, dried, and then put into methanol to be fixed for 5 min. The area of clear background and uniform distribution of spermatozoa was selected at low magnification. Areas with clear background, uniform sperm distribution and less overlapping and observed 400 sperms with complete structure under high magnification and counted the malformed sperms among them.

Sperm viability: The tail of the left epididymis was placed into 1 ml of preheated 37 °C balanced salt solution, clipped and blown to free the sperm; 10 μL of milky white liquid was added into 990 μL of preheated 37 °C balanced salt solution, and sperm motility and viability were detected by semen analyze.²⁰

Statistical analysis

All data are expressed as mean ± SD. Differences between groups were analyzed using Student’s t-test and Benjamini–Hochberg correction for multiple calibrations. Data were statistically processed using SPSS 20.0 software. P < .05 was considered a difference, and P < .01 was considered a statistically significant difference.

Results

MOO ameliorated CUMS-induced depressive-like behavior

The experiment was designed according to the steps in (Figure 1A). Four-week CUMS significantly reduced the rat body weight (P < .01). MOO significantly restored the CUMS-resulted decreased body weight at MOO-M, MOO-H, Flu in (Figure 1B). Low sucrose intake reflects an impaired reward response and anhedonia, one of the core symptoms of major depression. The CUMS-treated rats showed significantly decreased sucrose consumption (P < .01), and this effect was prevented in CUMS-exposed rats that received MOO (P = .005) in (Figure 1C). The FST can reflect the desperate behavior of animals, and the duration of immobility of the subject animals was used as the evaluation index, and the longer the duration of immobility the higher the degree of desperate behavior. Compared with the Control group, mice in the Model group had longer immobility time in FST, which was statistically different (P < .01) and showed longer duration of desperate behavior, and compared with the Model group, the immobility time of mice in the Fluoxetine group, the MOO-L, MOO-M, MOO-H decreased and was statistically different (P < .01), which indicated that the degree of desperate behavior was higher. Time was reduced and statistically different (P < .01), indicating that the administration of the drug reduced the frequency of the emergence of desperate behavior in mice. The experimental results are shown in (Figure 1D). The NSFT is often used to detect the anxiety level of animals, compared with the Control group, the mice in the Model group had a prolonged ingestion latency in NEST and showed a longer anxiety behavior, and there was a statistically significant difference (P < .01), and compared with the Model group, the latency of the mice in the Fluoxetine group and the MOO group was reduced and statistically significant difference (P < .01), and the experimental results are shown in (Figure 1E).

Figure 1.

Depressive-like behavior induced by CUMS and the protective effect of MOO. (A) Body weight. (B) Sucrose preference. (C) Forced swimming test. (D) Novelty-suppressed feeding test. ^#P < .05, ^##P < .01 vs the CON group; ^*P < .05, ^**P < .01 vs the CUMS group.

Effect of MOO on sexual behavior of mice

As shown in Table 2, MOO exhibited beneficial effect on the sexual behavior of mice. As compared to the control mice, MOO treatment shortened the capture and mount latency, increased capture, mount, ejaculation frequency. On the contrary, for the treatment with FLU, the capture and mount latency were increased, capture, mount, ejaculation frequency were declined.

Table 2.

Effects of MOO of sexual behavior of mice.

Unit/S	Capture latency	Capture frequency	Mount latency	Mount frequency	Ejaculation frequency
Control	100.32 ± 36.88	7.66 ± 3.41	250.91 ± 50.73	5.73 ± 1.22	2.74 ± 0.19
Model	265.38 ± 19.66	5.77 ± 2.60	308.38 ± 40.58	4.39 ± 1.05	1.44 ± 0.38
MOO-L	76.54 ± 5.42	9.50 ± 2.30	180.65 ± 30.42	6.73 ± 1.77	1.99 ± 0.10
MOO-M	68.33 ± 6.51	18.03 ± 4.03	130.33 ± 20.91	10.98 ± 2.44	2.54 ± 0.21
MOO-H	41.33 ± 5.77	21.33 ± 5.80	100.65 ± 15.79	11.41 ± 3.61	2.99 ± 0.21
Fluoxetine	140.65 ± 37.31	5.65 ± 3.90	140.65 ± 37.33	3.54 ± 0.98	1.66 ± 0.25

Effect of MOO on sexual organs of mice

Compared with Control group, Model control group mice organ coefficient are not significant (P > .05), did not find modeling on the mouse organ coefficient testicular coefficient, epididymal coefficient caused significant adverse effects. Compared with the Model, MOO dose groups on the mouse organ coefficient are not significant difference (P > .05), indicating that MOO on the mouse are no adverse effects (Table 3). Testicular tissue HE staining section results show that control group mice testicular tissue within the spermatogenic tubules arranged neatly and orderly, spermatogenic cells rich in number, good morphology rules; while the model group mice testicular weaving spermatogenic tubules within the spermatogenic cells are arranged loosely and disordered, the number of cells sharply reduced, the lumen of the tubule vacuolization serious. After the treatment of MOO the spermatogenic tubule cell arrangement is regular, uniform distribution, the cell number is obviously increased, the morphology is also obviously improved. It can be seen that MOO can slow down the testicular tissue structure damage caused by depression (Figure 2A). The results of HE staining section of epididymal tissue showed that spermatozoa in the epididymis of mice in the blank group were uniformly arranged, and almost no spermatogenic cells were shed in the lumen. Compared with the blank group, the sperm density in the epididymal ducts of the model group mice was significantly reduced, and a large number of detached spermatogenic cells were seen in the lumen. The sperm density in the epididymal ducts of the mice in the low, medium and high dose groups of MOO increased sequentially, and there was a significant decrease in the shedding of immature spermatogonial cells in the lumen of the ducts. This further verified that MOO could ameliorate the damage caused by depression and improve the spermatogenic function of the model mice (Figure 2B).

Table 3.

Effect of MOO on sexual organs of mice.

Groups	Number	Testes/weight (%)	Epididymis/weight (%)
Control	12	1.53 ± 0.12	0.21 ± 0.02
Model	12	1.52 ± 0.11	0.20 ± 0.01
MOO-L	12	1.51 ± 0.14	0.22 ± 0.03
MOO-M	12	1.52 ± 0.11	0.25 ± 0.02
MOO-H	12	1.55 ± 0.10	0.22 ± 0.04
Fluoxetine	12	1.50 ± 0.12	0.23 ± 0.01

Figure 2.

Sexual organs histopathologic changes. (A) Results of HE staining of testicular in mice (×100). (B) Results of HE staining of epididymis in mice (×100).

MOO improves sexual performance by ameliorating oxidative stress

The measurement of sex hormones is important for the study of reproductive system function, sexual development, sexual behavior and related diseases (McEwen & Milner, 2016). Compared with the Control group, the levels of LH in the serum of mice in the Model group increased significantly, with statistically significant differences (P < .01), while the levels of LH in the serum of mice in the Fluoxetine group, the MOO-L, MOO-M, MOO-H group decreased accordingly, with statistically significant differences (P < .01) compared with those in the Model group. Statistically different (P < .05 or P < .01). Compared with the Control group, the levels of FSH and T in the serum of mice in the Model group were significantly decreased, with statistical differences (P < .01), and compared with the Model group, the levels of FSH and T in the serum of mice in the Fluoxetine group, the MOO-L, MOO-M, MOO-H group were correspondingly increased, with statistically different (P < .05 or P < .01) (Figure 3A–C). Testicular marker enzymes mainly include LDH, ACP and ALP, the activities of testicular marker enzymes such as LDH, ALP, and ACP in the testicular tissues of the model group were significantly decreased (P < .01), which impaired the energy metabolism and supply of testicular tissues to some extent. Compared with the model group, medium and high doses of MOO significantly increased the enzyme activities of LDH, ALP and ACP in mouse testicular tissues (P < .001), which ameliorated the adverse effects on energy metabolism in mouse testicular tissues (Figure 3D–F). Oxidative stress is an important cause of testicular damage and hypogonadism in males. Depression-induced lipid peroxides MDA content was significantly higher than control group (P < .01). Superoxide dismutase, GSH, GSH-PX activities were significantly lower than control group (P < .01). After treatment with MOO, compared with the model group, the MDA content was significantly lower than that of the model group (P < .01), and the activities of SOD, GSH and GSH-PX were also significantly improved (P < .01). It suggests that barbiturates oligosaccharides can slow down depression-induced oxidative stress damage in testis (Table 4).

Figure 3.

MOO improves sexual performance by ameliorating oxidative stress. (A) Testosterone. (B) Luteinizing hormone. (C) Follicle-stimulating hormone. (D) Lactate dehydrogenase. (E) Acid phosphatase. (F) Alkaline phosphatase. ^#P < .05, ^##P < .01 vs the CON group; ^*P < .05, ^**P < .01 vs the CUMS group.

Table 4.

Effect of MOO on ameliorating oxidative of mice.

Groups	MDA (nmol/mgprot)	T-SOD (U/mgprot)	GSH (mg/gprot)	GSH-PX (μmol/L)
Control	0.43 ± 0.05	134.34 ± 7.32	0.55 ± 0.23	4.09 ± 0.82
Model	0.75 ± 0.32#	113.56 ± 8.78#	0.20 ± 0.01#	2.62 ± 0.27#
MOO-L	0.69 ± 0.10	120.38 ± 6.50*	0.43 ± 0.03*	3.42 ± 0.19
MOO-M	0.58 ± 0.06*	127.40 ± 4.85*	0.53 ± 0.21*	3.60 ± 0.20*
MOO-H	0.43 ± 0.05*	133.86 ± 10.45*	0.76 ± 0.31*	3.54 ± 0.30*
Fluoxetine	0.48 ± 0.10*	132.41 ± 15.83*	1.01 ± 0.09*	3.51 ± 0.36*

^# P < .05, vs the CON group; ^*P < .05, vs the CUMS group.

MOO protected against the neurodevelopmental deficits caused by CUMS

We used enzyme-linked immunosorbent assay to detect the expression of 5-HT, DA, NE and AChE in mice Hippocampal tissues. The assay results showed that MOO increased 5-HT, DA, NE and AChE levels in hippocampal tissues. Compared to CON, in hippocampal tissues, 5-HT, DA, NE and AChE levels reduced in MOD (P < .01) (Figure 4A–D).

Figure 4.

MOO protected against the neurodevelopmental deficits caused by CUMS. (A) Dopamine. (B) 5-Hydroxytryptamine. (C) Acetylcholinesterase. (D) Norepinephrine. ^#P < .05, ^##P < .01 vs the CON group; ^*P < .05, ^**P < .01 vs the CUMS group.

MOO improves sperm quality problems caused by CUMS

The mice sperm malformation test is an important method for assessing the genotoxicity of chemical substances. Compared with the Control group, the sperm malformation rate of mice in the Model group was increased and statistically different (P < .01). Compared with the Model group, the sperm malformation rate of mice in the MOO-L, MOO-M, MOO-H group was decreased and statistically different (P < .01), suggesting that the administration of MOO reduced the rate of sperm malformations in mice (Figure 5A). Sperm viability in mice is an important indicator of sperm fertilization ability and is directly related to the forward motility of sperm. Compared with the Control group, the sperm viability of mice in the Model group was reduced, and there was a statistical difference (P < .01). Compared with the Model group, the sperm viability of mice in the MOO-L, MOO-M, MOO-H group was increased and statistically different (P < .01) (Figure 5B). Mice sperm motility is also an important indicator of sperm fertilization ability, the sperm motility of mice in the Model group was decreased and statistically different (P < .01). Compared with the Model group, the sperm motility of mice in the MOO-L, MOO-M, MOO-H group was increased and statistically different (P < .01), indicating that the administration of MOO increased the sperm motility of mice (Figure 5C, D).

Figure 5.

MOO improves sperm quality problems caused by CUMS. (A) Sperm deformity. (B) Sperm motility. (C) Sperm forward. (D) Sperm oscillatory. ^#P < .05, ^##P < .01 vs the CON group; ^*P < .05, ^**P < .01 vs the CUMS group.

Discussion

We used the classical chronic unpredictable mild stress (CUMS) model to establish the depression model in male mice, and in this way to verify that MOO can improve the sexual dysfunction associated with depression. The results of this study showed that MOO not only exhibited antidepressant effects comparable to fluoxetine but also had a benign effect on sexual function and sexual ability in mice. In addition, the present study observed sexual dysfunction and decreased sexual performance, deteriorated sperm quality, altered testicular tissue morphology, disturbed serum hormone levels, and decreased testicular tissue marker enzyme activity in the depressed model group of mice, suggesting that the depressive state has a negative effect on the male reproductive system. In further experiments, we found that MOO was able to prevent and reduce the occurrence of the above abnormalities at certain doses. Specifically, MOO showed significant improvement in testicular organ index, sperm parameters, testicular tissue structure, reproductive hormone levels, testicular marker enzyme activities, and oxidative stress levels in mice. These findings provide new perspectives on the role of MOO in improving sexual function accompanying depression, and lay the foundation for an in-depth study of its mechanism.

Low mood, lack of pleasure, despair, and learning and memory loss are the main clinical symptoms of depression.²¹ Behavioral test is to evaluate the mouse behavioral state of the effective means of research, in the behavioral experimental results we found that MOO can increase the proportion of sugar and water consumption in mice, reduce the FST in the stationary time, increase ingestion latency in the NEST, through the above behavioral test results, we found that the MOO has good efficacy to improve the mice lack of pleasure, despair and learning and memory ability to reduce the performance of depression, suggesting that the MOO has antidepressant effect.²²

In the mating behavior, capture latency, capture frequency, mount latency, mount rate and ejaculation frequency were selected. The latency to capture and the number of capture in the mating test reflect the level of sexual desire of the mice; the latency to mount and mount rate of penetration reflect the process of ED; and the frequency to ejaculate corresponds to clinical premature ejaculation disease, and the problem of any one of these processes belongs to the problem of sexual dysfunction.^23,24 The mating test on mice showed that long-term depression caused delayed onset of sexual behavior and decreased the number of sexual acts, suggesting that depressed mice have sexual dysfunction. In the mating experiment, the latency to mount, latency to penetration and latency to ejaculation of depressed mice were significantly prolonged compared with the blank group, in addition, the number of mounts and the number of penetrations were reduced. MOO-L, MOO-M, MOO-H can significantly reduce the capture latency and ejaculation latency. MOO on the frequency of capture, mount number has increased, sexual behavior occurs latency time decreased, sexual behavior increased, suggesting that MOO in the treatment of male sexual dysfunction has good efficacy.

Spermatogenesis is a multistep process that may be interfered with by a variety of factors.²⁵ Destructive endogenous hormonal signals may affect the spermatogenesis process, which in turn may lead to a decrease in sperm count. T is essential for maintaining sperm viability and survival and also influences the physiological activities of reproductive organs.²⁶ The vast majority of T is synthesized and released by mesenchymal cells, and indirectly regulates spermatogenesis by acting on peritubular and supportive cells around the testis. Luteinizing hormone and FSH are considered to be the key factors in regulating testicular function.²⁷ Luteinizing hormone produces testosterone by stimulating testicular mesenchymal cells, and FSH and T regulate testicular mesenchymal cell activity through stimulation of testicular supportive cells, which promotes the proliferation and differentiation of germ cells.²⁸ In this study, the serum levels of LH were increased and the levels of T and FSH were decreased in the depression model mice. The treatment of MOO could normalize the serum levels of reproductive hormones and reduce the interference of reproductive hormone disorders on the spermatogenesis of mice.

Depression-induced changes in testicular enzyme activities in mice may be related to the impaired function of testicular support cells and metabolic disorders. Lactate dehydrogenase is a key enzyme involved in the process of glycolysis, and changes in its activity can reflect the altered metabolic state of tissues.²⁹ In testicular tissues, elevated LDH activity is usually associated with cellular damage, inflammatory response or metabolic disorders, and is one of the biomarkers of cellular damage. Changes in ACP activity may be related to sperm maturation and function, as well as to certain pathologies such as autoimmune diseases or infections.³⁰ Alkaline phosphatase is a membrane-bound enzyme that is widely distributed in tissues of the body.³¹ In testicular tissues, measurement of ALP activity can be used as an indicator of spermatogenic function and testicular developmental status. Changes in ALP activity may be associated with proliferation and differentiation of spermatogenic cells as well as with changes in sex hormone levels. In the present study, the decreased activities of LDH, ACP and ALP in depressed mice may have interfered with the energy supply of aerobic and anaerobic glycolytic processes, affecting the energy utilization for spermatogenesis in testicular tissues. In contrast, treatment with MOO increased the enzymatic activities of LDH, ACP and ALP, and may have further improved energy metabolism and energy supply for spermatogenesis in testicular tissues.

Toxicant-induced oxidative stress is thought to be closely related to male infertility.³² It is well known that oxidative stress leads to spermatogenic dysfunction by inducing peroxidative damage to the plasma membrane.³³ In the present study, we found that MDA levels were elevated and GSH, GSH-PX and SOD levels were decreased in testicular tissues of depressed mice. Since spermatozoa are rich in polyunsaturated fatty acids, they are more sensitive to oxidative damage compared to other cells. In the study in this chapter, depressed mice showed a significant decrease in sperm count and viability, and a significantly higher rate of sperm malformations. Histopathologic testing revealed a significant reduction in spermatogonia. The severe impairment of spermatozoa viability and quality in depressed mice was closely related to the apparent dysregulation of oxidative/antioxidant balance in the testis. The intervention treatment with MOO effectively reduced the deterioration of sperm quality, which may be related to the improvement of oxidative stress in the testicular microenvironment, as evidenced by the reduction of MDA levels and the increase of GSH, GSH-PX and SOD activities, suggesting that MOO have significant antioxidant effects in testicular tissues, including the enhancement of antioxidant enzyme activities and the reduction of lipid peroxidation.

Depression is an affective disorder in which monoamine neurotransmitters are involved in the regulation of emotional activity.³⁴ Dopamine, NE, AChE and 5-HT are all important components of the monoamine neurotransmitter machinery in depression. Among them AChE is a key enzyme that catalyzes the hydrolysis of acetylcholine (ACh), which is essential for the regulation of neurotransmission in the nervous system. AChE terminates neurotransmission mediated by the hydrolysis of ACh, a process that is critical for the maintenance of the normal function of the nervous system.³⁵ Norepinephrine is involved in the regulation of a variety of physiological functions of the body, such as the regulation of cardiovascular activity, mood, and body temperature. In addition, NE is also involved in the body’s memory and learning ability, and affects the body’s sleep and wakefulness, as well as the body’s perception of pain and anxiety. 5-Hydroxytryptamine is an inhibitory neurotransmitter, and its physiological action manifests itself in the form of inhibition of parasympathetic preganglionic fibers and excitation of sympathetic preganglionic fibers. This biological role can make its participation in a number of central nervous movement, such as mood control, but also can regulate the body’s sleep, and affect the body’s feeding behavior, but also affect the body’s anxiety and other negative emotions and the occurrence of sexual behavior. When the body is in a state of stress, the level of 5-HT will change, the above central nervous movement is also affected, and then depression and other mental illnesses occur.³⁶ Alterations in the DA mesolimbic reward system may lead to a lack of pleasure. Therefore, the expression levels of DA, 5-HT, AChE and NE are also specific indicators of depression. And 5-HT reuptake inhibitor and other antidepressant drugs can achieve antidepressant therapeutic effect by increasing the level of 5-HT, and the results of this experiment show that MOO can also achieve antidepressant therapeutic effect by increasing the content of DA, 5-HT, AChE, and NE.

Conclusion

Through a multifaceted mechanism of action, MOO not only show potential therapeutic effects on the improvement of depressive symptoms, but also shows a positive impact on the maintenance and improvement of mice reproductive health. Future studies will further explore the mechanism of the effect of MOO on depression and its concomitant sexual dysfunction, to provide a more solid scientific basis for its clinical application.

Funding

This project is supported by the Comparative study of accreditation systems for drug testing laboratories (2023MK215) and the phase I clinical and antidepressant effect mechanism study of Morinda officinalis oligosaccharides capsule (2014ZX09301307-014).

Conflicts of interest

None declared.