Application of dibutyl phthalate in construction of testis injury model

Abstract

The invention provides application of dibutyl phthalate in construction of a testis injury model. Animal experiments discover that dibutyl phthalate can stably construct a model of damaged testicular tissues of mice at the early stage of adolescence, and can destroy normal growth and development of the testis of the mice, destroy the testosterone secretion function of the testis of the mice, and destroy the blood testosterone barrier and the normal structure of the testicular tissues of the mice. The invention develops new application of dibutyl phthalate, provides a new choice for constructing a mouse testis injury model at the early stage of adolescence, and is beneficial to related research of reproductive diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the construction of animal models, specifically the application of dibutyl phthalate in the construction of a testicular injury model. Background Technology

[0002] Male infertility is a global health problem, accounting for approximately 20-70% of all infertility cases (Agarwal A, Mulgund A, Hamada A, et al. A unique view on male infertility around the globe[J]. Reproductive biology and endocrinology, 2015, 13: 1-9.). The testes are the primary male reproductive organs, and their main functions are spermatogenesis and stimulating the growth and development of reproductive organs. Impaired testicular function is a significant cause of male infertility. Because testicular dysfunction is a latent and long-term process, damage can only be diagnosed when pathological and physiological changes occur. This is especially true when testicular development is disrupted during embryonic or fetal gonadal development, directly causing male reproductive dysfunction. However, the lack of specific and sensitive animal models for detecting testicular damage limits medical research on this condition. Summary of the Invention

[0003] In order to address the problems in the prior art, the present invention aims to provide the application of dibutyl phthalate in constructing a testicular injury model.

[0004] The present invention adopts the following technical solution: Application of dibutyl phthalate in constructing a testicular injury model.

[0005] Dibutyl phthalate (DBP) is a common phthalate plasticizer, mostly a colorless to pale yellow oily liquid. It is commonly used in PVC (polyvinyl chloride) plasticizing, and as a plasticizer or additive in some resins, rubbers, coatings / inks, etc. Its chemical formula is C2. 16 H 22 O4, the structural formula is as follows: .

[0006] Furthermore, the model is an animal model. Even further, the model is a pre-pubescent mouse testicular injury model.

[0007] In one embodiment of the present invention, the application is to disrupt the normal developmental function of the testes.

[0008] In one embodiment of the present invention, the application is to disrupt the normal secretory function of the testes.

[0009] In one embodiment of the invention, the application is to disrupt the structural integrity of testicular tissue.

[0010] In one embodiment of the invention, the application is to disrupt the integrity of the blood-testis barrier in testicular tissue.

[0011] This invention also provides a method for constructing a male testicular injury model induced by dibutyl phthalate.

[0012] A method for constructing a male testicular injury model induced by dibutyl phthalate includes the following steps: selecting pre-pubertal BALB / c male mice as experimental mice; administering dibutyl phthalate by gavage once daily from day 22 to day 35 after birth to induce a male testicular injury model, with a dibutyl phthalate dose of 100-500 mg / kg / day.

[0013] The inventors used the testicular organ coefficient to detect the effect of dibutyl phthalate (DBP) on the normal growth and development of mouse testes. By measuring testicular weight and corresponding mouse body weight, the study showed that DBP significantly delayed and disrupted the normal growth and development of mouse testes compared to the control group. The effect of DBP on testosterone secretion in mouse testes was detected using ELISA. The results showed that the serum testosterone concentration in mice exposed to DBP was significantly lower than that in the control group. HE staining was used to detect the effect of DBP on the structure of mouse testicular tissue. The results indicated that the testicular tissue structure of the control group was normal, while the seminiferous epithelium structure of mice exposed to DBP was disordered, with spermatogenic cells sloughing off and significant vacuolation and cleft formation. Biotin tracer assay, Western blot, and immunohistochemistry were used to detect the effects of dibutyl phthalate on the blood-testis barrier structure in mice. The results showed that, compared with the control group, biotin in mice exposed to dibutyl phthalate could cross the blood-testis barrier, the expression of blood-testis barrier-related proteins was reduced, and the expression of cytoskeleton-related proteins shifted to an unstable direction, indicating that the integrity of the blood-testis barrier was disrupted and its function was impaired. Beneficial effects

[0014] This invention provides the application of dibutyl phthalate (DBP) in constructing a testicular injury model. Animal experiments have shown that DBP can stably construct a prepubertal mouse testicular tissue damage model. It can disrupt normal testicular growth and development, impair testosterone secretion, and damage the blood-testis barrier and normal testicular tissue structure. This invention expands the application of DBP, providing a new option for constructing a prepubertal mouse testicular injury model, which is beneficial for research on reproductive diseases. Attached Figure Description

[0015] Figure 1 This is a graph showing the statistical results of the testicular organ coefficients of mice in the model group and the control group; Figure 2 This is a graph showing the results of serum testosterone concentration detection in the model group and the control group mice; Figure 3 The images show the HE staining results of the testicular histological morphology of the model group and the control group mice. Figure 4 This is a graph showing the results of blood-testis barrier integrity testing in model and control mice; Figure 5 This is a graph showing the Wb detection results of blood-testis barrier-related proteins in the testicular tissues of mice in the model group and the control group; Figure 6 This is an image showing the IHC staining results of blood-testis barrier-related proteins in the testicular tissues of mice in the model group and the control group. Detailed Implementation

[0016] To further illustrate the objectives, technical solutions, and advantages of the present invention, the technical solutions of the present invention will be further described below. The following preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the following preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the scope defined by the claims of the present invention. Example 1

[0017] Establishment of a pre-pubertal BALB / c male mouse testicular injury model: All animal experimental procedures complied with the approval of the Animal Ethics Committee of Chongqing Medical University.

[0018] All male BALB / c mice in their pre-pubertal stage (postnatal day 21, PND 21) were purchased from Chengdu Yaokang Biotechnology Co., Ltd. and housed in the SPF animal facility of the Animal Center of the Second Affiliated Hospital of Chongqing Medical University. The housing conditions were 25±2℃, relative humidity 55±5%, and a 12 / 12 h diurnal cycle. BALB / c mice had free access to water and food, both provided by the animal facility of the Second Affiliated Hospital of Chongqing Medical University. Thirty male mice were randomly divided into three groups: a control group (corn oil), a D100 group (dibutyl phthalate 100 mg / kg / day), and a D500 group (dibutyl phthalate 500 mg / kg / day). From PND 22 to PND 35, the mice were administered corn oil once daily via gavage to the control group and dibutyl phthalate dissolved in corn oil to the treatment groups. If the blood-testis barrier integrity is to be tested, 2 mg / kg CdCl2 is injected intraperitoneally into the control mice 48 hours in advance as a positive control. 30 minutes before sampling, 50 μl of Sulfo-NHS-LC-Biotin solution (10 mg / ml, prepared with PBS containing 1 mM CaCl2) is injected into the interstitium of one testis of all mice selected for blood-testis barrier integrity testing, and PBS is injected into the contralateral side as a control.

[0019] Experimental results: Testicular organ coefficients of model group and control group mice are shown in the figure. Figure 1 Serum testosterone concentrations in the model group and control group mice are shown in the figure. Figure 2 HE staining results of testicular tissue from model group and control group mice are shown in the figure. Figure 3 The results of the blood-testis barrier integrity test in the model group and control group mice are shown in the figure. Figure 4 The Wb detection results of blood-testis barrier-related proteins in testicular tissue of model group and control group mice are shown in the figure. Figure 5 The IHC staining results of blood-testis barrier-related proteins in the testicular tissues of the model group and control group mice are shown in the figure. Figure 6The experimental results showed that: ① The testicular organ coefficient of the model group mice was significantly reduced, indicating that dibutyl phthalate disrupted the normal growth and development of mouse testicular tissue; ② The serum testosterone concentration of the model group mice was significantly reduced; ③ The seminiferous epithelium structure of the control group was regular and the spermatogenic cells were tightly arranged, while the seminiferous epithelium structure of the testicular tissue of the model group mice was disordered, with spermatogenic cells detaching and obvious vacuoles and clefts forming; ④ The blood-testis barrier was intact in the negative control group, completely destroyed in the positive control group, and partially destroyed in the model group mice, allowing biotin to pass through the blood-testis barrier into the seminiferous tubules, with more significant destruction in the high-dose group than in the low-dose group; ⑤-⑥ Compared with the control group, the expression of blood-testis barrier-related linkers such as ZO-1, N-Cadherin, β-catenin, Occludin, and Connexin43 and the cytoskeletal protein Eps8 was reduced in the model group, while the expression of the cytoskeletal protein Arp3 was increased compensatorily, with more significant changes in the high-dose group. Immunohistochemical results showed that the positive signals of blood-testis barrier-related connective proteins such as ZO-1, N-Cadherin, β-catenin, Occludin, and Connexin 43, as well as the cytoskeletal protein Eps8, were reduced and more discontinuous in the testicular tissue of the model group mice, while the positive signal of the cytoskeletal protein Arp3 was slightly enhanced. This suggests that the exposure to dibutyl phthalate in the model group disrupted the stability of the cytoskeleton and intercellular connections in the testicular tissue and promoted compensatory cytoskeleton remodeling.

[0020] The above animal experiments demonstrate that dibutyl phthalate can stably construct a prepubertal mouse testicular tissue damage model. It disrupts normal testicular growth and development, impairs testosterone secretion, damages the blood-testis barrier, and alters the normal structure of mouse testicular tissue. Therefore, this invention expands the application of dibutyl phthalate, providing a new option for constructing a prepubertal mouse testicular injury model, which is beneficial for research on reproductive diseases and has significant implications.

Claims

1. Application of dibutyl phthalate in constructing a testicular injury model; the structural formula of the dibutyl phthalate is as follows: 。 2. The application as described in claim 1, characterized in that, The model is an animal model.

3. The application as described in claim 2, characterized in that, The model described is a pre-pubertal mouse testicular injury model.

4. The application as described in any one of claims 1-3, characterized in that, The application disrupts the normal developmental function of the testes.

5. The application as described in any one of claims 1-3, characterized in that, The application disrupts the normal secretory function of the testes.

6. The application as described in any one of claims 1-3, characterized in that, The application involves disrupting the structural integrity of testicular tissue.

7. The application as described in any one of claims 1-3, characterized in that, The application disrupts the integrity of the blood-testis barrier in testicular tissue.

8. A method for constructing a male testicular injury model induced by dibutyl phthalate, comprising the following steps: selecting pre-pubertal BALB / c male mice as experimental mice; administering dibutyl phthalate by gavage once daily from day 22 to day 35 after birth to induce a male testicular injury model, with the dibutyl phthalate dose being 100-500 mg / kg / day.

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