阅读说明：本技术 β-谷甾醇在缓解4-硝基苯酚所致生殖毒性中的应用 (Application of beta-sitosterol in relieving reproductive toxicity caused by 4-nitrophenol ) 是由 张永辉 刘冬梅 卢婷婷 李峰 于 2020-07-06 设计创作，主要内容包括：本发明公开了β-谷甾醇在缓解4-硝基苯酚所致生殖毒性中的应用,属于生物医药技术领域。本发明的研究表明,对于4-硝基苯酚所引起的TM3细胞形态损伤及凋亡现象,β-谷甾醇能够起到明显的保护作用,同时,β-谷甾醇对4-硝基苯酚暴露的TM3细胞睾酮分泌量具有显著的保护干预效果。本发明对于β-谷甾醇对4-硝基苯酚毒性的干扰作用及其作用机制的探索和研究,能够对人类预防及治疗4-硝基苯酚引起的雄性生殖疾病提供重要依据,研究具有实际意义。(The invention discloses application of beta-sitosterol in relieving reproductive toxicity caused by 4-nitrophenol, belonging to the technical field of biological medicines. The research of the invention shows that the beta-sitosterol can play an obvious role in protecting the morphological damage and apoptosis of TM3 cells caused by 4-nitrophenol, and meanwhile, the beta-sitosterol has an obvious protective intervention effect on the testosterone secretion of TM3 cells exposed by the 4-nitrophenol. The invention can provide important basis for human beings to prevent and treat male reproductive diseases caused by 4-nitrophenol by exploring and researching the interference effect of beta-sitosterol on the toxicity of 4-nitrophenol and the action mechanism thereof, and has practical significance for research.)

1. Application of beta-sitosterol in preparing a medicament for relieving animal reproductive toxicity caused by 4-nitrophenol.

2. Use according to claim 1, characterized in that: the reproductive toxicity refers to the reproductive toxicity of male animals.

3. Use according to claim 1, characterized in that: the reproductive toxicity refers to the reproductive toxicity of 4-nitrophenol on the reproductive cells of male animals.

4. Use according to claim 3, characterized in that: the male animal germ cells are TM3 cells.

5. Application of beta-sitosterol in preparation of medicines for relieving germ cell injury and/or germ cell apoptosis caused by 4-nitrophenol.

6. Application of beta-sitosterol in preparing a medicament for relieving the increase of germ cell ROS (reactive oxygen species) caused by 4-nitrophenol.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of beta-sitosterol in relieving reproductive toxicity caused by 4-nitrophenol.

Background

4-nitrophenol is a typical environmental endocrine disrupter and is widely applied to the manufacture of pesticides, bactericides, dyes, leather preservatives and medicines. The pollution of the 4-nitrophenol in the environment is mainly from industrial and agricultural production, automobile exhaust particulate matters and pesticide metabolites. 4-nitrophenol can easily enter the body through the skin, respiratory system, digestive system, etc. 4-nitrophenol is not easy to biodegrade and has a biological enrichment effect, and low-dose 4-nitrophenol can produce stronger toxic effect on human beings at the top of a food chain through the enrichment of the food chain.

The endocrine disrupting effects of 4-nitrophenol are mainly manifested by estrogen-like activity and antiandrogen activity, and can disturb the steroid hormone signaling pathway of the body, with reproductive toxicity. The existing research on the reproductive toxicity of the 4-nitrophenol mainly focuses on the observation and monitoring of toxicity indexes, and the research on the action mechanism of the toxicity indexes is less, so that the research on the detoxification method of the 4-nitrophenol is slow. The industrial detoxification method of 4-nitrophenol is mainly physical adsorption or chemical degradation, and the biodegradation method of 4-nitrophenol has attracted more and more attention in recent years due to the problems of residual adsorbent, toxicity of chemical degradation products and the like. Plant extracts are increasingly regarded as a safer food additive.

Beta-sitosterol is a natural active substance similar to a cyclic alcohol structure in plants, is an important component of plant cell membranes, is also a precursor for synthesizing various hormones, vitamin D and steroid compounds, and most of plant foods contain a certain amount of beta-sitosterol, wherein the beta-sitosterol content in plant oil, seeds, nuts, grains and beans is particularly rich, and is one of end products of plant metabolism and widely exists in roots, stems, leaves, fruits and seeds of the plants in the forms of free fatty acid esters, glycosides and the like. The beta-sitosterol which is ingested by human beings and animals in daily life has the effects of reducing blood fat, reducing the risk of cardiovascular and cerebrovascular diseases, resisting inflammation, resisting oxidation, improving immunity, resisting cancer, regulating growth, regulating hormone-like functions and the like, wherein the research on the aspects of inhibiting cholesterol absorption, reducing the level of total cholesterol in blood plasma and the risk of cardiovascular and cerebrovascular diseases is deep, but the application of the beta-sitosterol as the intervention agent for the male reproduction toxicity of the 4-nitrophenol is not reported.

Disclosure of Invention

In order to solve the defects of the existing 4-nitrophenol hazard mechanism research and prevention and control measures, the invention provides a new application of beta-sitosterol, namely the intervention effect of the beta-sitosterol on the reproduction toxicity of mouse testicular interstitial cells TM3 exposed by the 4-nitrophenol. According to the result of in vitro experiments, the invention discovers that the beta-sitosterol has a good relieving effect on germ cell injury caused by 4-nitrophenol when being applied to the experiment of 4-nitrophenol germ cells.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of beta-sitosterol in preparing a medicament for relieving animal reproductive toxicity caused by 4-nitrophenol.

Further, the reproductive toxicity refers to reproductive toxicity of male animals.

Further, the reproductive toxicity refers to the reproductive toxicity of 4-nitrophenol on male germ cells.

Further preferably, the male animal germ cells are TM3 cells.

Application of beta-sitosterol in preparation of medicines for relieving germ cell injury and/or germ cell apoptosis caused by 4-nitrophenol.

Application of beta-sitosterol in preparing a medicament for relieving the increase of germ cell ROS (reactive oxygen species) caused by 4-nitrophenol.

The application of the beta-sitosterol in the preparation of the medicaments is within the protection scope of the invention.

The research of the invention shows that the beta-sitosterol can play an obvious role in protecting the morphological damage and apoptosis of TM3 cells caused by 4-nitrophenol, and meanwhile, the beta-sitosterol has an obvious protective intervention effect on the testosterone secretion of TM3 cells exposed by the 4-nitrophenol.

Drawings

FIG. 1 is a schematic diagram of the experimental technique of the present invention.

FIG. 2 shows the cell activity rates of example 1 at various concentrations of β -sitosterol and 4-nitrophenol.

FIGS. 3 and 4 Effect of beta-sitosterol and 4-nitrophenol on apoptosis in example 2.

FIG. 5 is a graph of ROS levels 24h after stimulation with β -sitosterol and 4-nitrophenol in example 3.

FIGS. 6 and 7 show the expression levels of key genes in the testosterone synthetic pathway after 24h of action of β -sitosterol and 4-nitrophenol in example 4.

In the above drawings: control is a Control group, PNP is a 4-nitrophenol group, and PNP + beta-sitosterol is 4-nitrophenol + beta-sitosterol group.

Detailed Description

The method firstly adopts an MTT method to detect whether the beta-sitosterol has an inhibiting effect on the reproductive toxicity of the 4-nitrophenol and the concentration gradient playing the inhibiting effect, and finally determines the appropriate concentration gradient. Further research was subsequently conducted, including: chemiluminescence (CL) to detect intracellular levels of Reactive Oxygen Species (ROS); detecting apoptosis by a Flow Cytometer (FCM); RT-PCR method is used to detect mRNA expression quantity of genes such as StAR and 3 beta-HSD which play a role in regulating and controlling testosterone synthesis.

MTT results show that in a few concentrations which are determined preliminarily, when the concentration of the beta-sitosterol is 10-20 mu M/L, the intervention effect on the reproductive toxicity of the 4-nitrophenol is shown, and the damage of the 4-nitrophenol on the cell activity can be obviously reduced. The result of the apoptosis rate shows that the beta-sitosterol can play an obvious role in protecting the apoptosis phenomenon caused by 4-nitrophenol. The ROS detection result shows that the beta-sitosterol can play a certain protective intervention role in the ROS change of TM3 cells caused by 4-nitrophenol. According to the RT-PCR result, mRNA expression levels of StAR and 3 beta-HSD playing a key role in a testosterone synthetic pathway of a TM3 cell are detected, the StAR and 3 beta-HSD levels of a 4-nitrophenol group are both remarkably reduced, and compared with the 4-nitrophenol group, mRNA expression levels of StAR and 3 beta-HSD for 10 and 20 mu mol/L beta-sitosterol dry prognosis are both remarkably increased.

From the above experimental results, the inventors concluded the following: the beta-sitosterol has an inhibiting effect on the apoptosis of TM3 cells exposed by 4-nitrophenol; beta-sitosterol may inhibit cells by reducing ROS levels in 4-nitrophenol-exposed TM3 cells; beta-sitosterol can improve the expression of key regulatory proteins StAR and 3 beta-HSDmRNA in a testosterone synthetic pathway of a TM3 cell exposed by 4-nitrophenol. In a word, the beta-sitosterol has better nutritional intervention and protection effects on functional damages such as reduction of activity of TM3 cells exposed by 4-nitrophenol, apoptosis, increase of ROS and the like.

The research result of the invention has certain significance for understanding the function of the beta-sitosterol and playing the mechanism of reproductive toxicity intervention function and the like. The beta-sitosterol not only has a health-care function, but also has important prevention and treatment effects on the harm of some toxic substances to organisms, provides important basis for the exploration and research on the interference effect of the beta-sitosterol on the toxicity of the 4-nitrophenol and the action mechanism thereof, can prevent and treat male reproductive diseases caused by the 4-nitrophenol, and has practical significance for the research.

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

The research technical route of the invention is shown in the attached figure 1. The invention preselects beta-sitosterol with stronger oxidation resistance as a target object and researches the possible detoxification pathway and mechanism of the beta-sitosterol to the injury of TM3 cells exposed by 4-nitrophenol.

The invention adopts a mouse testicular interstitial cell line TM3 as an experimental model, carries out toxicity molding on cells at a concentration (10 mu M/L) which is relatively obvious in inhibition effect of 4-nitrophenol on the activity of TM3 cells and is explored through experiments, then screens the proper concentration of beta-sitosterol through an MTT method, and observes the change of cell morphology and size by a microscope. Further, the intervention effect and the specific mechanism of the toxic effect of the beta-sitosterol on the 4-nitrophenol are discussed by researching the ROS level change of the TM3 cells and the like. Finally, the expression quantity of mRNA such as StAR, 3 beta-HSD and the like in the synthesis process of testosterone is detected by an RT-PCR method.

In the following examples, all measurements were expressed as mean ± standard error (X ± SE), and the data were statistically analyzed using Graphpad Prism5 software, with p < 0.05 indicating statistical significance.