Zhenyuan, a kidney- and spleen-tonifying Chinese medicine compound granule, improves erectile dysfunction and fatigue in orchiectomized rats

Introduction

In middle-aged and elderly populations, there is a notable decline in human physiological functions and hormone levels, which gives rise to a series of health problems, such as erectile dysfunction (ED). ED is defined as the persistent inability to obtain and maintain an adequate penile erection for satisfactory sexual intercourse. Age is a well-known risk factor for ED. The prevalence of ED among men without any comorbidities increases from 10% to 79% from the age of 40 and 80 years (1). In addition, the risk of ED increases sharply with comorbidities (e.g., diabetes, hypertension, obesity, and cardiovascular disease) (1,2). It is estimated that 40% of men aged 40–70 years worldwide suffer from ED, significantly impacting the quality of life and psychosocial health of men and their partners (3).

The early treatment options for ED, such as psychotherapy, penile prostheses, intracavernosal injection, and intraurethral therapy, have limited effectiveness and applicability. The introduction of oral phosphodiesterase type 5 inhibitors (PDE5Is) in the late 1990s revolutionized the treatment of ED and rapidly established itself as a first-line monotherapy (4). These drugs bind to the catalytic subunit of phosphodiesterase type 5 (PDE5) and inhibit the inactivation of cyclic guanosine monophosphate (cGMP), thus reducing intracellular free Ca₂⁺ and resulting in greater relaxation of cavernous smooth muscle (5). Currently, four PDE5Is, i.e., sildenafil, vardenafil, tadalafil, and avanafil, are globally available (6,7). Several new PDE5Is are available only in specific countries [mirodenafil and udenafil in Korea (8,9), lodenafil in Brazil (10), and aildenafil in China (11)]. Although PDE5Is are highly effective for at least 60% of all cases of ED, safety concerns remain, especially in patients with hypertension, hypotension, arrhythmia, unstable angina pectoris, congestive heart failure, or myocardial infarction. Furthermore, the use of PDE5Is is absolutely contraindicated in patients taking nitrate or nitrate-donors preparations (5).

ED belongs to the category of ‘yinwei’ and ‘impotence’ in traditional Chinese medicine (TCM). TCM has a long history of understanding and treating this disease. Among the treatments, the most widely used are Chinese herbal compound formulas and acupuncture therapy (12). TCM formulas, such as Shuganyiyang capsule, Yougui pill, Xiaoyao pill, etc., have been increasingly used as complementary and alternative medicine treatments for ED patients, and their efficacy has been confirmed by more and more studies (12-15). Zhenyuan granule (ZYKL) used in this study is composed of the water extracts of Epimedium (Yin-Yang-Huo), Panax ginseng (Ren-Shen), Glycyrrhiza uralensis (Gan-Cao), Poria cocos (Fu-Ling), Zingiberis rhizoma (Gan-Jiang), and Salvia miltiorrhiza (Dan-Shen). It was modified from Aikeqing formula, a TCM formula developed by our research group for human immunodeficiency virus/acquired immune deficiency syndrome (AIDS) patients. Clinically, it enhances immune function and quality of life, and alleviates the side effects of anti-AIDS medications by tonifying the kidney and the spleen (16). We have also previously reported that Aikeqing prevented ovariectomy-induced bone loss in rats (17). In ZYKL, Epimedium has the effect of warming kidney-yang (18), and Panax ginseng, Glycyrrhiza uralensis, and Poria cocos can invigorate the Spleen and replenish Qi (19-21). Kidney and spleen are the two primary organs related to ED in TCM understanding. The kidney stores essence and dominates reproduction. The kidney-qi is an important physiological basis for erectile function. The spleen is the foundation of acquired constitution and the biochemical source of Qi and blood, and promotes the erectile function by enhancing kidney-yang. Additionally, Salvia miltiorrhiza is added to the formula to promote blood circulation. We speculate that ZYKL tonifies kidney, strengthens spleen, and invigorates blood circulation, therefore, it could be used in the treatment of ED. In this study, we investigated the sexual stimulation and anti-fatigue effects of ZYKL on orchiectomized rats and the related underlying mechanisms. We present this article in accordance with the ARRIVE reporting checklist (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-311/rc).

Methods

Drug preparation

The TCM decoction pieces, including Epimedium sagittatum (Sieb. et Zucc.) Maxim., Panax ginseng C. A. Mey., Glycyrrhiza uralensis Fisch., Poria cocos (Schw.) Wolf., Zingiber officinale Rosc., and Salvia miltiorrhiza Bge., were provided by Yangming Herbal Materials Company (Kunming, China), and authenticated by the corresponding author (Y.J.H.). All the TCM decoction pieces met the quality control criteria of the Chinese Pharmacopeia (2020 Edition) (22).

A total of 36.0 kg of mixed TCM decoction pieces were prepared in a mass ratio of 6:3:3:2:2:2, and decocted in pure water at a ratio of 15 times of the amount of decoction pieces for 2 h, with this process repeated 3 times. The extraction solution was combined and filtered. The filtrate was concentrated, dried in vacuum, crushed and granulated, a yield of 17.0%. The high-performance liquid chromatographic fingerprints of ZYKL were shown in Figure 1A.

Figure 1 Effects of ZYKL on food intake and body weight. (A) High-performance liquid chromatographic fingerprints of reference compounds (red) and ZYKL (blue) used for quality control of ZYKL. 1, liquiritin; 2, ginsenosides Rb3; 3, epmedin A; 4, epmedin B; 5, salvianolic acid C; 6, epmedin C; 7, icariin; 8, quercetin; 9, glycyrrhetinic acid; 10, tanshinone IIA. (B) Food intake. (C) Body weight. All data are presented as the mean ± standard deviation. **, P<0.01, significantly different from the Sham group. ^#, P<0.05; ^##, P<0.01, significantly different from the ORX group. ORX, orchiectomy; TP, testosterone propionate; ZYKL, Zhenyuan granule; ZYKL-H, ZYKL high-dose; ZYKL-L, ZYKL low-dose; ZYKL-M, ZYKL middle-dose.

Animals and experimental procedure

Male Sprague-Dawley rats (specific pathogen free grade, 4–5 weeks old and weighing 150±10 g) were purchased from Guangdong Medical Laboratory Animal Center [license No. SCXY (Yue) 2022-0002] and kept at 20–25 ℃ and 50–70% relative humidity with a 12-h light-dark cycle and free access to food and water. One week after adaptive feeding, the rats were anesthetized with 1% isoflurane, and bilateral testis were surgically removed (23). Another 10 rats were used as the sham-operated group (Sham) only underwent laparotomy without testicular removal. After operation, five rats in one cage; 20,000 units/kg of penicillin were injected subcutaneously for 3 consecutive days. Subsequently, the rats with orchiectomy (ORX) were randomized into the following 5 groups (n=10 per group): ORX, testosterone propionate (TP, 2 mg/kg), ZYKL low-dose (ZYKL-L, 0.94 g/kg), ZYKL middle-dose (ZYKL-M, 3.76 g/kg), and ZYKL high-dose (ZYKL-H, 7.52 g/kg). The low dose for rat was calculated based on the normal adult dose (9 g/d) and the body weight (BW) ratio between adult and rat. The middle and high doses were defined as four and eight times the low dose, respectively. The TP group was given intramuscular injection, and the other groups were given oral administration (20 mL/kg) once a day for 21 days. The sham and ORX group were given the equal volume normal saline solution.

All procedures employed were approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine (No. 20220907002), in compliance with Chinese institutional guidelines for the care and use of animals. No rat deaths were observed during the experiment.

Sexual behavior assessment

Sexual behavior parameters observed in this study included erection latency (EL), mount latency (ML), and mount frequency (MF). After 21 days of administration, percutaneous electrostimulation of the perineum was performed using an electric stimulator with 4 mA current. The time (in seconds) from stimulus to penis erection was recorded as EL (24). The mounting behavior tests were conducted in a quiet and dimly lit room. The test male rat was placed in a 50 cm × 50 cm × 40 cm cage, and after an adaptation period of 5 min, a stimulus-receptive female rat, which had been subcutaneously injected with estradiol benzoate (0.1 mg/kg) and progesterone (2 mg/kg) 24 and 4 h prior to the experiment, respectively, was introduced into the cage. ML (seconds from introduction of the female to the occurrence of the first mount) and MF (the number of mounts) within 20 min were recorded. If mount did not occur within 20 min, ML was marked 1,200 s (25).

Weight-loaded forced swimming (WLFS) test

WLFS test was performed as described earlier, with minor modifications (26). Rats were placed separately in a cylindrical tank (height 82 cm × diameter 65 cm) filled with water (25±1 ℃), and lead wire (5% of BW) was attached to the tail root of each rat. The exhaustive swimming time was recorded when the rats remained submerged for 10 s without surfacing.

Blood lactic acid (BLA) and blood urea nitrogen (BUN) measurements

The rats swam for 10 min (30±1 ℃) without weight bearing. Blood was collected by retro-orbital puncture before swimming, 0 min and 20 min after swimming, and BLA levels were measured using a lactic acid assay kit (Cat No. A019-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The area under the curve (AUC) of BLA levels was calculated using GraphPad Prism 6.01 (GraphPad Software). The serum was separated and the concentration of BUN was detected using a BUN assay kit (Cat No. C013-2-1; Nanjing Jiancheng Bioengineering Institute).

Serum hormone and nitric oxide synthase (NOS), nitric oxide (NO), and cGMP assays

At the end of the experiment, the mice were fasted overnight, and anesthetized with 1% isoflurane, and blood was collected from the aortaventralis. After centrifugation at 3,000 r/min for 10 min, the supernatant was collected as serum and stored at −20 ℃ until analysis. The concentrations of testosterone (T), NO, and cGMP, and the activity of NOS were quantified using a rat T or cGMP enzyme-linked immunosorbent assay kit (Cat No. ml059506 and ml003133; Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China), and an NO or NOS assay kit (Cat No. A012-1 and A014-2; Nanjing Jiancheng Bioengineering Institute), respectively.

Histopathological examination

After blood collection, the rats were sacrificed by exsanguination under isoflurane anesthesia. The penis was removed, weighed, fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sliced into 3–4-µm sections (27). After hematoxylin and eosin (H&E) and Masson staining, all sections were scanned using a digital pathology slides scanner (KF-PRO-005, Konfoong Bioinformation Tech Co., Ltd., Ningbo, China) and evaluated.

Statistical analysis

SPSS software (version 24.0; SPSS, Inc., IBM Corp., Armonk, NY, USA) was used for the statistical analysis. Results were expressed as the mean ± standard deviation. Comparisons between groups were analyzed via one-way analysis of variance with post hoc Tukey’s test, and P<0.05 was considered statistically significant.

Results

Effects of ZYKL on food intake and BW

During the experiment, the average food intake of the orchiectomized rats was lower than that of the Sham group. However, at the end of the experiment, there was no significant difference in weight gain between the Sham group and the ORX group. Compared to the ORX group, rats administered with TP exhibited a significant increase in food consumption and BW. The ZYKL-treated rats showed no significant alterations in food intake and BW, except for weight gain in the medium-dose group (see Figure 1B,1C).

Effects of ZYKL on the erectile response and mounting behavior in ORX rats

As shown in Figure 2A-2C, ORX led to a significant reduction in erectile function and sexual activity. Compared to the Sham group, EL and ML in the ORX group were significantly prolonged, and MF was significantly decreased. In ZYKL-H group, EL and ML were 81% and 83% shorter than ORX group, respectively, and MF was significantly higher than ORX group. Compared to the ORX group, the treatment with middle dose of ZYKL led to a significant decrease in EL, and the low dose of ZYKL slight improved EL, ML, and MF, but the difference was not statistically significant.

Figure 2 Effects of ZYKL on hormone levels and sexual behaviors in ORX rats. (A) Erection latency. (B) Mount latency. (C) Mount frequency. (D) Total testosterone levels. All data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01, significantly different from the Sham group. ^#, P<0.05; ^##, P<0.01, significantly different from the ORX group. ORX, orchiectomy; TP, testosterone propionate; ZYKL, Zhenyuan granule; ZYKL-H, ZYKL high-dose; ZYKL-L, ZYKL low-dose; ZYKL-M, ZYKL middle-dose.

Effects of ZYKL on hormone levels and histological morphology in ORX rats

Compared to the sham-operated rats, the serum T levels of the orchiectomized rats significantly decreased. The administration of ZYKL resulted in a significant dose-dependent increase in T levels when compared to the untreated rats (see Figure 2D).

The histological examination of the penis by H&E and Masson staining was shown in Figure 3. H&E staining revealed that trabeculae were evenly distributed in the corpus cavernosum of the sham-operated rats, with abundant blood sinuses, and some red blood cells were visible in the sinuses. The penile weight and diameter of ORX group were obviously smaller than those of Sham group, and the number of blood sinuses and small vessels were significantly reduced. The administration of TP effectively reversed the morphological and pathological alterations of the penis induced by ORX. Although there was no significant difference in penile diameter and weight between the ZYKL groups and the untreated group, the number of blood sinuses and small vessels increased significantly, especially in the high-dose group (see Figure 3A,3B).

Figure 3 Histological examination of penis. (A) Penile weight. (B) Representative H&E staining. Scale bars: 20 µm. (C) Representative Masson staining. Scale bars: 40 µm. (D) Quantitative analysis of collagen fibers. All data are presented as the mean ± standard deviation. **, P<0.01, significantly different from the Sham group. ^#, P<0.05; ^##, P<0.01, significantly different from the ORX group. BW, body weight; H&E, hematoxylin and eosin; ORX, orchiectomy; TP, testosterone propionate; ZYKL, Zhenyuan granule; ZYKL-H, ZYKL high-dose; ZYKL-L, ZYKL low-dose; ZYKL-M, ZYKL middle-dose.

The degree of penile fibrosis was also observed by Masson staining. The results showed that the area of collagen fibers (in blue) was significantly increased in the orchiectomized rats, and this was reversed after ZYKL and TP treatment (see Figure 3C,3D).

Effects of ZYKL on the NOS-NO-cGMP signaling pathway in ORX rats

As shown in Figure 4, serum NOS activity, NO and cGMP concentrations in the orchiectomized rats were markedly decreased compared with the sham-operated rats. ZYKL treatment significantly reversed the decrease in serum NO, cGMP levels and NOS activity induced by ORX.

Figure 4 Effects of ZYKL on the NOS-NO-cGMP signaling pathways. (A) NOS activity. (B) NO levels. (C) cGMP levels. All data are presented as the mean ± standard deviation. **, P<0.01, significantly different from the Sham group. ^#, P<0.05; ^##, P<0.01, significantly different from the ORX group. cGMP, cyclic guanosine monophosphate; NO, nitric oxide; NOS, nitric oxide synthase; ORX, orchiectomy; TP, testosterone propionate; ZYKL, Zhenyuan granule; ZYKL-H, ZYKL high-dose; ZYKL-L, ZYKL low-dose; ZYKL-M, ZYKL middle-dose.

Anti-fatigue effects of ZYKL in ORX rats

In the WLFS test, there was no significant difference in exhaustive swimming time between Sham, ORX, TP, and ZYKL groups. However, the levels of BLA and BUN were significantly higher in the ORX group than in the Sham group after non-weight-bearing swimming. TP treatment had no significant effect on BLA and BUN levels in orchiectomized rats. However, ZYKL treatment significantly reduced the concentration of BLA and BUN (see Figure 5).

Figure 5 Anti-fatigue effects of ZYKL. (A) WLFS test. (B) BUN levels. (C) BLA levels. (D) AUC-BLA. All data are presented as the mean ± standard deviation. *, P<0.05; **, P<0.01, significantly different from the Sham group. ^#, P<0.05; ^##, P<0.01, significantly different from the ORX group. AUC, area under the curve; BLA, blood lactic acid; BUN, blood urea nitrogen; ORX, orchiectomy; TP, testosterone propionate; WLFS, weight-loaded forced swimming; ZYKL, Zhenyuan granule; ZYKL-H, ZYKL high-dose; ZYKL-L, ZYKL low-dose; ZYKL-M, ZYKL middle-dose.

Discussion

ZYKL is a TCM formula, which has the therapeutic properties of invigorating kidney and spleen and promoting blood circulation. Some of herbs in the formula, such as Epimedium and Panax ginseng, have shown promising efficacy in treating ED (14). In this study, we assessed the improved effects of ZYKL on sexual function in an orchiectomized rat model. TP was used as the positive control. T is the predominant androgen in males. An increasing evidence substantiates the critical role of T in erectile function. T deficiency is associated with decreased erectile function and T levels are inversely associated with the severity of ED (28,29). In our animal model, a significant decline in T levels and sexual function was observed after ORX, which was restored by TP supplementation. Similar to TP, ZYKL increased T levels, shorten the erectile latency induced by electrical stimulation, improved the mounting behaviors, and reversed pathological alterations of penile tissue (including increasing the number of blood sinuses and small vessels, and reducing fibrosis) in orchiectomized rats. T is produced primarily in testicular Leydig cells, and is partially converted from precursors (predominantly dehydroepiandrosterone-sulfate) synthesized by the adrenal cortex (30). Some Chinese herbs for tonifying kidney, such as Ginseng and Epimedium, may enhance T levels by regulating adrenal function (31,32). Moreover, we found that the activity of NOS and the concentrations of NO and cGMP increased after ZYKL treatment. NO, synthesized by the oxidation of L-arginine catalyzed by NOS, acts as the primary mediator of penile erection. It activates soluble guanylate cyclase, leading to an increase in cGMP concentration, which promotes cavernosal relaxation and vasodilation (33). cGMP’s effects can be terminated by PDE5, which breaks down its phosphodiester bond. PDE5Is are widely recognized as oral therapeutics for ED by inhibiting the degradation of cGMP mediated by PDE5. To date, more than ten varieties of Chinese medicinal herbs used in the treatment of ED have been proved to have the effect of activating the NO-cGMP signaling pathway (34). Especially, ginsenosides, the main active ingredient of ginseng, enhanced NO release and cGMP accumulation in corpus cavernosum both in vivo and in vitro (14,35,36). The water extract of Epimedium and icariside II (a flavonoid derived from Epimedium), increases the cellular cGMP by enhancing the expression and activity of NOS (37-40). In present study, our data showed that ZYKL increased total T, NO, and cGMP concentrations and NOS activity, suggesting that ZKKL may improve ED by activating hormone secretion and NOS-NO-cGMP signaling pathway. Interestingly, T and NOS-NO-cGMP signaling pathway share an interdependent relationship. T enhances NO-cGMP signaling pathway by upregulating the expression of NOS (41), and NOS-NO-cGMP signaling pathway, in turn, facilitates T synthesis (42).

In addition, we found that the levels of BLA and BUN after swimming in the ZYKL-treated groups were significantly lower than those in the ORX group. Lactic acid is produced during exercise as a product of glycolysis and lowers the pH of blood and muscles, leading to fatigue and tissue damage (43). With the increase of muscle fatigue in the human body, the level of BLA increases (44). Urea nitrogen is the metabolic outcome of protein and amino acids. In the case of muscle fatigue, the degradation of proteins results in a temporary increase in urea nitrogen levels (45). Therefore, BLA and BUN are two crucial biochemical parameters related to fatigue (44-47). Previous studies have revealed that some Chinese herbs in ZYKL formula, such as Panax ginseng (48-50), Epimedium (51), and Salvia miltiorrhiza (52-54), possess anti-fatigue properties. It is not surprising that ZYKL was shown to relieve fatigue in this study.

Conclusions

In conclusion, ZYKL, a kidney- and spleen-tonifying Chinese medicine compound granule, has the potential to improve ED by elevating T levels and activating NOS-NO-cGMP signaling pathway, and relieve fatigue by reducing BLA and BUN levels. In ZYKL formula, Glycyrrhiza uralensis, Poria cocos, and Zingiberis rhizoma are both medicinal and edible materials, while Epimedium, Panax ginseng, and Salvia miltiorrhiza can be utilized as health supplements (http://www.nhc.gov.cn/zwgk/wtwj/201304/e33435ce0d894051b15490aa3219cdc4.shtml). Therefore, ZYKL may be developed as a new Chinese medicine or functional food for improving ED and anti-fatigue. However, the ORX model used in this study differs from the spleen-kidney deficiency model, and the results cannot fully represent the effect of ZYKL on ED caused by spleen-kidney deficiency. Moreover, there are many causes of ED, and the indications for ZYKL require further clarification.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-311/rc

Data Sharing Statement: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-311/dss

Peer Review File: Available at https://tau.amegroups.com/article/view/10.21037/tau-2025-311/prf

Funding: This work was supported by the National Key Research and Development Program of China (No. 2019YFE0109800).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2025-311/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures employed were approved by the Experimental Animal Ethics Committee of Guangzhou University of Chinese Medicine (No. 20220907002), in compliance with Chinese institutional guidelines for the care and use of animals.

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