ZHANG Ya-guang, WANG Quan-sheng, HUANG Zi-yan, Chen Lu, SONG Ji-xiang,LIN Zi-li

1. Graduate School of Guangxi University of Chinese Medicine, Nanning 530001, China

2. Guangxi Key Laboratory of Molecular Biology of Preventive Medicine of Traditional Chinese Medicine, Nanning 530023, China

3. The First Affiliated Hospital of Guangxi University of Chinese Medicine, Nanning 530023, China

Keywords:Xuduan Zhongzi prescription Oligoasthenospermia Heat shock protein Apoptosis

ABSTRACT Objective: To investigate the effect of Xuduan Zhongzi prescription on the expression of HSP60 and HSP90 in testicular spermatogenic cells and semen quality of oligozoospermia and asthenospermia model rats. Methods: A total of 40 SPF male rats were randomly divided into 10 blank control group and 30 model group. After successful modeling, the model group was divided into model control group, levocarnitine group and Xuduan Zhongzi prescription group with 10 rats in each group. The rats in levocarnitine group and Xuduan Zhongzi prescription group were given corresponding drugs by gavage according to the equivalent measurement of humanbody, the blank control group and model control group were given the same amount of normal saline by gavage, and the rats in each group were killed after 8 weeks of continuous administration. Take the left epididymis of each group for sperm quality detection, take the left testis of each group, observe the histomorphological changes by HE staining, and detect the expression of HSP60 and HSP90 in testicular tissue by immunohistochemistry and Western blot. Results: HE staining showed that compared with the blank control group, the convoluted tubules in the model group were significantly atrophic, degenerated and irregularly arranged,the spermatogenic epithelium showed a large number of vacuoles, Sertoli cell degeneration,obvious inflammatory infiltration in stromal cells, stromal cell proliferation, scattered rows of epithelial cells, a large number of exfoliation and degeneration, and sperm were rare. After the intervention of Xuduan Zhongzi prescription, the above lesions were significantly improved,the structure of seminiferous tubules was relatively regular and orderly, denser than that of the model group, sertoli cells and stromal cells increased significantly, the seminiferous epithelium was thicker than that of the model group, with rich layers, and a large number of sperm could be seen in the lumen. Immunohistochemistry and Western Blot showed that the expression of HSP60 and HSP90 increased significantly after the intervention of Xuduan Zhongzi prescription in model rats (P＜0.01). The sperm quality test results showed that compared with the blank control group, the sperm concentration and activity rate in the model group decreased significantly, (P＜0.01). After continuous seed cutting, the sperm concentration and activity rate of model rats were significantly increased, (P＜0.01). Conclusion: Xuduan Zhongzi prescription may inhibit the apoptosis of testicular spermatogenic cells in oligoasthenospermia model rats by increasing the expression of HSP60 and HSP90, so as to improve sperm quality.

1. Introduction

Oligospermia is the main cause of male infertility, accounting for 75% of male infertility [1], according to the World Health Organization (WHO) estimates, about 9% of the world's couples have fertility disorders, among which males Factors account for 50% [2]. According to WHO's 5th edition "Laboratory Manual for Human Semen Examination and Processing", the total number of sperm in one ejaculate is less than 39×106/mL, or the sperm concentration is less than 15×106/mL, which is diagnosed as oligospermia, and the percentage of sperm with forward motility is less than Thirty-two percent were diagnosed with asthenozoospermia[3]. The number of patients with oligoasthenozoospermia continues to increase, and it also shows an increasing trend year by year [4].According to relevant analysis, the concentration and motility of male sperm have continued to decline in recent years, which also means that male fertility is declining [5]. The common causes of oligoasthenozoospermia include male reproductive system infection,varicocele, cryptorchidism, and endocrine factors [6].

Heat shock proteins (HSPs) are a class of chaperone molecules present in cells [7]. Under physiological conditions and stress states,HSPs can be used as molecular chaperones to participate in the folding, assembly, transport and deletion of cellular proteins. Error protein [8]. HSP plays an important role in male reproduction.In the HSP family, HSP60 is involved in testis development and spermatogenesis, and HSP90 can regulate spermatogenic cell division and spermatogenesis and protect sperm from oxidative damage [9, 10]. Preliminary studies have shown that cutting off the seeds can inhibit the apoptosis of spermatogenic cells and improve the repair ability of spermatogenic cells after oxidative damage,thereby improving sperm quality and promoting spermatogenesis[11, 12], but the underlying mechanism remains to be further studied and improved. Therefore, based on the analysis of HSP60,HSP90 expression and sperm quality in testis tissue of rats with oligoasthenospermia, this study explored part of the mechanism of action of Xuduan Zhongzi prescription in the treatment of oligoasthenospermia.

2. Materials and methods

2.1 Experimental animals and groups

40 SD male rats, SPF grade, weighing 200-220 g, purchased from Hunan Slike Jingda Laboratory Animal Co., Ltd., animal license[SCXK (Xiang) 2019-0004], this experiment was approved by the Animal Ethics Committee of Guangxi University of Traditional Chinese Medicine approve. After adaptive feeding for 1 week,10 animals were randomly selected as blank control group. After successful modeling, the remaining 30 animals were divided into model control group, L-carnitine group, and Xuduan Zhongzi group according to the random number table method, with 10 animals in each group.

2.2 Test Drugs

Cyclophosphamide powder injection (Baxter company, import drug registration number: H20160467); L-carnitine oral liquid 10mL: 1 g(Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical Co., Ltd., H19990372); Xuduan Zhongzi prescription(The composition of the medicine: 15 g each of eucommia, dodder,medlar, privet, Rhizoma Drynariae, Achyranthes, Codonopsis,Atractylodes, and Huai Yam) (Chinese medicine without decoction,produced by Jiangyin Tianjiang Pharmaceutical Co., Ltd., Jiangsu Province) , Drug License No.: 1203002).

2.3 Main reagents and instruments

HSP60 primary antibody, Affinity company, batch number:AF0184; HSP90 primary antibody, batch number: Ab203126,purchased from Abcam company in the United States. Sperm quality detection system (Beijing Weili New Century Technology Development Co., Ltd.); semi-automatic rotary microtome(Thermoshandon Finesse E); paraffin embedding machine (Leica EG1150H+C); inverted phase contrast microscope (Olympus BX43),desktop refrigerated centrifuge (Eppendrof 5430R).

2.4 Modeling and drug administration

The oligoasthenospermia model was prepared according to reference [13]. Except for the blank group, the other rats were injected with 40 mg·kg-1·d-1cyclophosphamide by intraperitoneal injection for 1 week. After successful modeling, the traditional Chinese medicine group was given the equivalent dose of the drug into an aqueous solution for intragastric administration, given 10 g crude drug dose /(kg-1·d-1) Xuduan Zhongzi prescription, and the western medicine group was given L-carnitine oral liquid 0.1 g·kg-1·d-1, the normal group and the model group were given the same amount of normal saline every day for 8 consecutive weeks.

2.5 Semen Analysis

24 h after the last administration, the experimental rats were anesthetized by intraperitoneal injection of 5% chloral hydrate, the left epididymis of the rats was quickly removed, and the adipose tissue and fascia were removed, the blood was washed with normal saline, weighed and placed in 2 mL Cut into pieces and mix well in a plate of normal saline, incubate in a constant temperature water bath at 37 ℃ for 20 minutes, shake gently, and use sperm according to the WHO "Laboratory Inspection Manual for Human Semen Examination and Processing" standard parameters Counting plates were used to detect sperm concentration and sperm motility.

2.6 HE detection

The rat testis tissue from each group was taken and placed in 10%neutral formalin for 24 hours, then routinely embedded in paraffin,serially sectioned by a paraffin microtome, routinely deparaffinized,stained with hematoxylin and eosin (HE), and then mounted. The morphological changes were observed with an optical microscope and photographed.

2.7 Immunohistochemical detection

Take the paraffin-embedded sections of testicular tissue, and carry out the general immunohistochemical method in strict accordance with the instructions of the reagents. Under normal circumstances,the cytoplasm and nucleus of positive cells HSP60 and HSP90 can be stained yellow or brown after staining. Image Pro Plus software was used to analyze the immunohistochemical pictures of positive cells HSP60 and HSP90, and the area of positive cells was calculated.

2.8 Western Blot detection

Take the testis tissue and grind it in liquid nitrogen, add protein lysing solution, and lyse it in ice water for 0.5 h-1 h, fully lyse it with an ultrasonic pulverizer, repeat 5 times; Protein loading buffer,boiled for 10 min, electrophoresed, transferred to membrane,blocked, added primary antibody and incubated overnight at 4 ℃,placed in TBST for shaking and washing, added secondary antibody and incubated for 1 h, washed membrane with TBST, and finally added chemiluminescence dropwise on the membrane The solution was exposed and imaged in the imaging system, and the protein bands were analyzed using Image-Pro Plus software.

2.9 Statistical processing

IBM SPSS Statistics 25 software was used for relevant data analysis, data were expressed as(±s), one-way analysis of variance was used for comparison between groups, LSD test was used for comparison between two groups, and GraphPad Prism 9.0 software was used to draw statistical graphs, P＜0.05 was expressed The difference was statistically significant.

3. Results

3.1 General performance

During the experiment, the mental activity, diet, body weight, and coat color of the rats in the blank control group were all normal;after modeling, the rats in each group showed apathy, diet reduction,weight loss, dull coat color, and unresponsiveness to varying degrees.After 1 week of drug intervention, the above symptoms improved.During the experimental administration, a total of 3 rats died (1 in the model control group, 1 in the L-carnitine group, and 1 in the Xuduan Zhongzi prescription group), and the remaining 37 rats were finally included in this study.

3.2 Comparison of sperm quality

After testing, compared with the blank control group, the sperm concentration and motility of the other groups were significantly decreased (P＜0.01), among which the model group had a small number of scattered sperm and low motility rate; The sperm concentration and motility rate of the Ting group and the Xuduan Zhongzi prescription group were significantly increased (P＜0.01);there was no significant difference between the L-carnitine group and the Xuduanzifang group (P＞0.05), and there was no statistical significance. See Table 1.

Tab 1 Semen quality comparison of rats in each group (±s)

Tab 1 Semen quality comparison of rats in each group (±s)

Note: Compared with the blank group,ΔΔP＜0.01, compared with the model control group, ##P＜0.01

Group nSperm concentration(×106 /mL)Sperm motility(%)Blank control group 10 50.35±3.14 61.62±3.66 Model control group 9 14.10±2.97ΔΔ 15.49±2.63ΔΔ L-carnitine group 9 34.43±2.89## 36.45±2.52##Xuduan Zhongzi prescription group 9 36.47±3.12## 39.50±3.74##F 235.60 330.09 P 0.000 0.000

3.3 Histomorphological analysis of testis in each group of rats

Under the light microscope, the testicular convoluted tubules of the rats in the blank control group were round or oval, with regular distribution, without atrophy or degeneration. The seminiferous epithelium was thick, and the spermatogenic cells were rich in layers, neatly arranged and dense, and visible in the lumen. Lots of sperm. In the model control group, the convoluted tubules were obviously atrophied, degenerated and irregularly arranged, the seminiferous epithelium was vacuolated, Sertoli cells degenerated,the inflammatory infiltration in the interstitial cells was obvious,the interstitial cells were proliferated, and the epithelial cells were scattered and shed in large quantities. Degeneration, sperm are rare. Compared with the model control group, the lesions above the L-carnitine group and the Xuduan Zhongzi prescription group were significantly improved, the seminiferous tubules were relatively regular in structure, arranged in an orderly manner, and denser than the model group. Compared with the model group, the seminiferous epithelium was thicker and richer in layers, and a large number of sperms could be seen in the lumen. See Figure 1.

3.4 Immunohistochemical detection and analysis of rats in each group

After staining, the cytoplasm and nucleus of positive cells HSP60 and HSP90 could be stained yellow or brown. Compared with the blank control group, the expression of HSP60 and HSP90 in the model control group was significantly reduced (Figure 2B, Figure 3B); Compared with the model group, the expressions of HSP60 and HSP90 in the Xuduan Zhongzi prescription group were significantly increased after drug intervention (Figure 2C, Figure 2D; Figure 3C,Figure 3D).

Fig 1 Pathological results of testis in each group(HE, 200×)

Fig 2 Expression of HSP60 in testis of rats in each group(IHC, 200×)

Fig 3 Expression of HSP90 in testis of rats in each group (IHC, 200×)

3.5 Western Blot analysis of rats in each group

Compared with the blank control group, the expression levels of HSP60 and HSP90 in the testis tissue of the model control group were decreased (P＜0.01). The expression of L-carnitine group and Xuduan Zhongzi prescription group were not significantly different(P＞0.05), and there was no statistical significance. See Table 2 and Figure 4.

Fig 4 Expression of HSP60 and HSP90 in testis (Western Blot)

Tab 2 Expression of HSP90 and HSP90 in testicular tissue of rats in each group (±s)

Tab 2 Expression of HSP90 and HSP90 in testicular tissue of rats in each group (±s)

Note: Compared with the blank group,ΔΔP＜0.01, compared with the model control group, ##P＜0.01

HSP90 expression level Blank control group 10 0.73±0.05 1.06±0.05 Model control group 9 0.53±0.02ΔΔ 0.59±0.08ΔΔ L-carnitine group 9 0.82±0.02## 0.85±0.05##Xuduan Zhongzi prescription group 9 0.88±0.07## 0.87±0.06##F 83.25 86.58 P 0.000 0.000 Group n HSP60 expression level

4. Discussion

Oligoasthenospermia is the main cause of male infertility, and the main clinical manifestations are decreased sperm count and sperm motility. Common causes of oligoasthenozoospermia include male reproductive system infection, endocrine factors, varicocele,cryptorchidism, etc. [14]. Clinically, western medicine mainly treats oligoasthenozoospermia based on the cause, mostly estrogen receptor antagonists, L-carnitine, antioxidants, etc., but the curative effect is not exact and there are many adverse reactions in the process of treatment, which often have in clinical application. Limitations [15,16]. There is no oligoasthenospermia in Chinese medicine. According to its clinical symptoms, it is generally considered to be in the categories of oligozoospermia, thinness, and infertility. Traditional Chinese medicine has accumulated rich experience in the process of treatment, and has the advantages of low cost and few side effects.According to related researches, the effect of traditional Chinese medicine compound in improving sperm quality is better than that of chemical drugs [17].

Chinese medicine believes that oligoasthenozoospermia is closely related to the liver, spleen and kidney, mainly due to kidney deficiency, and also has liver stagnation, spleen deficiency, blood stasis, and damp-heat, spleen, blood circulation, dampness, etc. [18].In the course of clinical and scientific research, our research group has applied the continuum seed prescription, which is generalpurpose and nourishing, and has indeed achieved good curative effects in clinical treatment [19, 20]. "Cultivation of seeds" originated from "Medical Zhengyin" written by Yue Fujia, a physician in the Ming Dynasty. The combination of Xueduan and ginseng in the prescription can invigorate the kidney, invigorate the spleen,invigorate the blood and generate sperm; the combination of Dodder,Lycium barbarum and Ligustrum lucidum can nourish the liver and kidney, benefit the essence and blood; Achyranthes, Eucommia,Rhizoma Drynariae, and Red Teng can invigorate the kidney and generate sperm, and promote blood circulation. communication.Modern pharmacological studies have shown that serotonin has an antioxidant effect [21]; icariin in epimedium can increase the thickness of seminiferous tubules and sperm count, and epimedium alkaloids can improve animal libido and can significantly increase sperm motility and viability Eucommia ulmoides and Achyranthes can inhibit the oxidative stress and apoptosis of rat testis, thereby improving sperm quality [23, 24]. Both dodder and wolfberry have

the effect of improving reproductive function [25]. Lycium barbarum polysaccharide in wolfberry can significantly repair damaged spermatogenic cells, and the dodder flavonoids contained in dodder can promote the function of the hypothalamus-pituitary gonadal axis and inhibit spermatogenesis. Epithelial cell apoptosis, and also has the effect of anti-oxidative damage [26-28]. The combination of various medicines has the effect of invigorating the kidney and spleen, promoting blood circulation, promoting proficient and dredging collaterals.

HSP60 is a highly conserved chaperone protein with bidirectional pro-apoptotic and anti-apoptotic regulatory roles [29]. In the process of spermatogonia mitosis, the expression of HSP60 is significantly up-regulated, which also shows that the mitogenic activity of spermatogonia is correlated with the expression of HSP60. At the same time, related studies also show that the lower the expression level of HSP60 in the testis, the higher the spermatogenesis ability.Poor [30-32]. HSP90 is abundant in cells, accounting for 1%-2% of the total cellular protein content, and is involved in spermatogenesis and capacitation. Relevant studies have shown that HSP90 deletion can cause sperm meiotic arrest and testicular atrophy [33-36]. The results of this study showed that after drug intervention in the model rats, the results of HE staining showed that compared with the blank control group, the convoluted tubules of the rats in the model group were significantly atrophied, degenerated and irregularly arranged,and the seminiferous epithelium showed a large number of vacuoles.Sertoli cell degeneration, obvious inflammatory infiltration in interstitial cells, proliferation of interstitial cells, scattered epithelial cells, a large number of shedding degeneration, rare sperm. The above lesions were significantly improved after the intervention of Xuduan Zhongzi prescription. The structure of seminiferous tubules was relatively regular and arranged in an orderly manner,which was denser than that of the model group. Sertoli cells and stromal cells were significantly increased. A large number of sperm can be seen in the cavity. Immunohistochemistry and Western Blot detection showed that the expression levels of HSP60 and HSP90 were significantly increased in model rats after the intervention of Xuduan Zhongzi prescription (P＜0.01). The results of sperm quality test showed that compared with the blank control group,the sperm concentration of the model group and motility rate,after the intervention of Xuduan Zhongzi prescription, the sperm concentration and motility rate of model rats were significantly improved(P＜0.01). And the sperm concentration and the activity rate were significantly increased(P＜0.01).

In conclusion, the mechanism of Xuduan Zhongzi prescription promoting spermatogenesis in oligoasthenospermia model rats may be through up-regulating the expression of HSP60 and HSP90,thereby inhibiting the apoptosis of spermatogenic cells, improving the spermatogenesis microenvironment and sperm quality. The underlying mechanism remains to be further studied.

Author’s contribution:

Zhang Yaguang: index detection, data analysis, writing papers;Huang Ziyan: model establishment, index detection; Chen Lu: model establishment, index detection; Song Jixiang: model establishment,material collection; Lin Zili: model establishment and material collection; Wang Quansheng: general researcher people, instructors.All authors declare that there is no conflict of interest.