Efficacy And Mechanism Of Cistanches Herba Extract in Treating Reproductive Dysfunction in Rats With Kidney-Yang Deficiency Based On Metabolomics Ⅱ
Feb 06, 2025
3.2 Effect of Cistanche extract on the general state of rats with kidney yang deficiency
Before the experiment, rats in each group were in good spirits, with normal hair and normal eating and drinking. During the experiment, no abnormalities were found in the normal group rats, and their body weight increased steadily with the extension of feeding time; as the adenine modeling time prolonged, the model group rats' hair fell off and became rough, their spirits were low, their reactions were slow, they curled up and arched their backs, their urine volume increased, their body weight decreased significantly, and their rectal temperature decreased significantly (P < 0.001), showing obvious symptoms of kidney yang deficiency; Cistanche extract and LC intervention treatment can alleviate the above symptoms to varying degrees, among which the rectal temperature was significantly adjusted (P < 0.001), see Figure 1.
Fig.1 Effect of Cistanches Herba extract on the body weight and rectal temperature of rats with kidney-Yang deficiency(𝑥̅±s(n=10(
3.3 Effect of Cistanche extract on the visceral-brain ratio coefficient of rats with kidney-yang deficiency
Compared with the normal group, the visceral-brain ratio coefficients of the testis and epididymis in the model group were significantly reduced (P < 0.001); compared with the model group, the visceral-brain ratio coefficients of the testis and epididymis in each dose group of Cistanche extract were significantly increased (P < 0.05, P < 0.01, P < 0.001), and the visceral-brain ratio coefficients of the testis and epididymis in the LC group were also significantly increased (P < 0.01, P < 0.001), see Figure 2.
Table 2 Effect of Cistanches Herba extract on the sperm motility parameters in rats with kidney-Yang deficiency(𝑥̅±s,n=10)
3.5 Effect of Cistanche extract on sperm count, motility and deformity rate in rats with kidney yang deficiency
Normal sperm has a sickle-shaped head, a slender body, a naturally curved tail, and a smooth overall shape. The morphology of other deformed sperm is shown in Figure 3. Compared with the normal group, the sperm density and sperm motility of the rats in the model group were significantly reduced (P<0.01, P<0.001), and the sperm deformity rate was significantly increased (P<0.001); compared with the model group, the sperm density and motility of the rats in each dose group of Cistanche extract and the LC group were significantly increased (P<0.05, P<0.01, P<0.001), and the sperm deformity rate was significantly reduced (P<0.001), as shown in Figure 4.
3.6 Effect of Cistanche extract on testicular and epididymal tissue morphology in rats with kidney yang deficiency
The testicular seminiferous tubules of rats in the normal group were intact, with regular arrangement of spermatogenic cells at all levels and clear layers. Abundant spermatocytes and elongated spermatids were visible in the lumen. Compared with the normal group, the proportion of lesioned seminiferous tubules in rats in the model group increased significantly (P<0.001), which was manifested as irregular structure of seminiferous tubules, thinning of tubular walls, enlargement of lumen, increased number of exfoliated cell blocks in the lumen, decreased number of linear sperm, and disordered arrangement of spermatogenic cells. Compared with the model group, the proportion of testicular seminiferous tubule lesions in rats in all dose groups of Cistanche extract and LC group was significantly reduced (P<0.05, P<0.01), among which the low dose group of Cistanche extract had better improvement effect, which was equivalent to the LC group, as shown in Figure 5.
Get more details about cistanche for improving sexual function
The epididymal tubules of rats in the normal group were intact, the epithelial cells were neatly arranged, and evenly distributed sperm could be observed; the epididymal tubules of rats in the model group were swollen, the epithelial cells were disorderly arranged, and the number of sperm in the lumen was significantly reduced; compared with the model group, the epididymal lumen lesions of rats in the Cistanche extract groups and the LC group were significantly improved, the epithelial cells were tightly and neatly arranged, and the number of normal sperm in the lumen increased and was evenly distributed, as shown in Figure 6.
4 Discussion
Kidney Yang deficiency syndrome is a syndrome type in traditional Chinese medicine, which refers to the deficiency of kidney Yang and the inability of the body to warm up. Modern medicine believes that kidney Yang deficiency is related to dysfunction of the hypothalamic-pituitary target gland (mainly adrenal glands, gonads and thyroid gland) axis [11]. Adenine can act on the HPG axis of rats, causing its dysfunction, thereby interfering with the homeostasis of the body's hormone levels, showing symptoms similar to kidney Yang deficiency. T level can be used as one of the detection indicators of this model [12]. The other general diagnostic criteria for kidney Yang deficiency are applied to model rats, namely, decreased body temperature, weight loss, curling up, sparse hair, increased urine volume, loose stools, etc. [9,13]. Cistanche deserticola is one of the most commonly used drugs in traditional Chinese medicine prescriptions for tonifying kidney Yang. Its key active substance, phenylethanoid glycosides, has also been reported to have the functions of regulating HPG axis homeostasis and anti-fatigue [14]. In this study, rats with adenine deficiency were used as a model of kidney yang deficiency. The improvement effect of Cistanche deserticola extract on rats with kidney yang deficiency was evaluated from the general manifestations of rats and the changes in hormones related to the HPG axis. The results showed that all doses of Cistanche deserticola extract could improve the symptoms of kidney yang deficiency in rats caused by adenine and reduce the HPG axis inhibitory effect of adenine.
Oligospermia and asthenospermia can be classified into the categories of "childlessness", "infertility", "cold sperm" and "sperm blockage" in traditional Chinese medicine. The main pathogenesis includes kidney yang deficiency, damp heat, yin deficiency and hyperactivity of fire, qi stagnation and blood stasis, among which kidney yang deficiency is the most common [15-16]. Kidney yang deficiency cannot warm the sperm palace, resulting in cold sperm and qi, thus childlessness [17]. The efficacy of warming and tonifying kidney yang prescriptions for kidney yang deficiency and infertility has also been widely reported [18]. The results of this experiment showed that the extracts of Cistanche deserticola at all doses, a traditional Chinese medicine for invigorating kidney yang, could significantly alleviate the decrease in the ratio of the testicular and epididymal organs to the brain caused by adenine; the results of sperm dynamics parameter analysis showed that the extracts of Cistanche deserticola at all doses could significantly improve the sperm dynamics parameters including VCL and VAP, and significantly increase sperm motility and sperm concentration; the results of sperm fluorescence showed that the extracts of Cistanche deserticola could significantly improve the decrease in sperm motility caused by adenine; the results of HE staining showed that the extracts of Cistanche deserticola could significantly alleviate the sperm deformity caused by adenine, and alleviate the morphological changes of the testis and epididymis caused by adenine; the results of ELISA showed that the extracts of Cistanche deserticola significantly adjusted the serum level of T, which is necessary for testicular spermatogenesis, and the low-dose extracts of Cistanche deserticola showed better results. The above results show that the extracts of Cistanche deserticola have an improving effect on the reproductive damage of rats with kidney yang deficiency.
LC, also known as L-carnitine, is an essential energy metabolism substance in the human body. It usually exists in the male reproductive system in two forms: free and acetylated. It provides energy for the movement and maturation of sperm in the epididymis by promoting the oxidation of fatty acids and is widely used in the treatment of male reproductive system diseases[19]. Studies have shown that LC can alleviate the HPG axis disorder caused by glutamine, regulate the levels of luteinizing hormone and follicle-stimulating hormone, and increase the T level by regulating the expression levels of genes such as cytochrome P450 family 11subfamily A member 1 (CYP11A1), steroidogenic acute regulatory protein (STAR), etc., which are key enzymes in T synthesis[20]. In addition, LC can remove excessive reactive oxygen species in the testis caused by varicocele, reduce oxidative stress damage, and thus restore testicular spermatogenesis[21]. In this experiment, LC was selected as a positive control. The results showed that LC could effectively alleviate the reproductive damage of rats caused by adenine, which was consistent with the previous research results; and the Cistanche deserticola extract showed a therapeutic effect comparable to LC.
Metabolomics can qualitatively and quantitatively analyze small molecule metabolites in the body as a whole, and then combine the information integration of multiple databases for multidimensional statistical analysis, which can characterize the complex relationship between physiology, pathology and drug action [22-23]. Based on LC-MS technology, this experiment conducted metabolomics analysis on serum samples of rats in the normal group, model group, and low-dose Cistanche deserticola group, aiming to explore the metabolic mechanism of Cistanche deserticola in intervening in reproductive damage caused by adenine in rats with kidney yang deficiency. The results showed that a total of 22 related differential metabolites and 3 metabolic pathways were screened. Cistanche deserticola extract mainly affects the levels of 13-HpODE, 20-carboxy-arachidonic acid (20-COOH-AA), LPC (O-18:0/0:0), pantethine, and bimatoprost through metabolic pathways such as lipid metabolism, pantothenic acid and coenzyme A biosynthesis, thereby exerting a reproductive protective effect.
Lipids play a vital role in signal transduction, energy production, and maintaining cell homeostasis[24]. Lipid molecules can be divided into eight categories based on their structures: fatty acids, glycerolipids, phospholipids, sphingolipids, sterols, pre-alcohols, glycolipids, and polyketides. Disorders in fatty acid metabolism are closely related to abnormal sperm capacitation[25] and chronic kidney damage[26]. 13-HpODE and 20-COOH-AA are metabolites of linoleic acid and arachidonic acid, respectively, that participate in lipid peroxidation reactions. When their levels increase, it indicates that the body is more sensitive to oxidative stress. This may lead to excessive production of reactive oxygen species, which in turn leads to decreased male reproductive function[27]. LPC is the most abundant type of lysoglycerophospholipid in human blood and can induce oxidative stress in testicular cells, causing reproductive damage[28]. Some researchers have found that LPC (O-18:0/0:0) induces stress and apoptosis of renal tissue cells by activating the expression of peroxisome proliferator activated receptor delta (PPARδ) in the renal tubules, suggesting that it may be one of the biomarkers of renal function deterioration [29]. It suggests that Cistanche deserticola extract may play a reproductive protective role in rats with kidney yang deficiency by downregulating lipid peroxidation levels and inhibiting oxidative stress. Pantothenic acid has an important effect on testosterone secretion and sperm motility. Its metabolite pantethine is the raw material for the synthesis of coenzyme A, which further participates in the regulation of energy metabolism and fatty acid metabolism [30]. Cistanche deserticola extract may promote the body's energy metabolism and reduce the damage of oxidative stress to the reproductive system of rats with kidney yang deficiency by adjusting the level of pantethine. This indicates that Cistanche deserticola extract may play an antioxidant role by regulating lipid metabolism, the biosynthesis of pantothenic acid and coenzyme A, and thus protect the reproductive function of rats with kidney yang deficiency.
In addition, Leydig cells are the main site of T production, and the formation of T precursor pregnenolone depends on mitochondrial function in Leydig cells. Studies have shown that excessive accumulation of reactive oxygen species in Leydig cells is associated with T synthesis disorders. Excessive reactive oxygen species induce mitochondrial damage, resulting in the inhibition of adenosine triphosphate (ATP) and adenosine cyclophosphate (cAMP) synthesis, thereby inhibiting the expression of T synthesis-related enzymes and leading to a decrease in T levels [31]. The results of this experiment showed that Cistanche deserticola extract significantly reversed the adenine-induced decrease in serum T levels. The results of metabolomics analysis were similar to those reported in the literature, that is, Cistanche deserticola extract may promote T synthesis by alleviating Leydig cell oxidative damage.
The results of the content determination of Cistanche deserticola extract showed that echinacoside, verbascoside, salidroside, betaine, and cistancheside A may be potential active ingredients. Previous studies have shown that echinacoside can protect the testis from oxidative stress damage by activating antioxidant enzymes and mitogen-activated protein kinase (MAPK) signaling-related factors [32]; salidroside can upregulate the body's antioxidant factors to counteract the damage of the blood-testis barrier caused by oxidative stress, thereby promoting spermatogenesis [33]; verbascoside can significantly enhance the proliferation activity of hydrogen peroxide-treated TM3 cells, partially protecting them from oxidative stress damage [34]; betaine can upregulate the expression levels of antioxidant-related genes in the testis of lead-exposed mice, including superoxide dismutase 1, glutathione peroxidase 1, and catalase, and significantly improve the testicular Johnson score [35]; cistancheside A has also been reported to reduce the abnormal increase of reactive oxygen species in spermatogonia induced by hypoxia and reduce cell apoptosis [36]. All these suggest that echinacoside, verbascoside, salidroside, betaine, and cistancheside A may be the important material basis for Cistanche to alleviate the symptoms of kidney yang deficiency in rats induced by adenine. In summary, Cistanche extract can alleviate the symptoms of kidney yang deficiency in rats induced by adenine, as well as the pathological damage of the testis and epididymis, restore the damaged HPG axis function, improve the reduced sperm quality, and thus improve the reproductive function of rats with kidney yang deficiency; its mechanism of action may be related to the regulation of lipid metabolism, biosynthesis of pantothenic acid and coenzyme A, etc. Echinacoside, verbascoside, salidroside, betaine, and cistancheside A are the possible potential bioactive material basis, which needs further experimental verification.
