CN103749457B - Application of dequalinium chloride as mouse birth control medicine - Google Patents

Abstract

The invention relates to the application of dequalinium chloride as a birth control drug for rodents. It is found through research that CTBS is a unique sperm-egg binding receptor on rodent sperm, and its inhibitor dequalinium chloride can effectively inhibit the birth control of mice. Its half-inhibition constant is less than 100nM, and it has little toxic effect on human body; it is also found that CTBS is expressed on the sperm of rodents, and the CTBS antibody can inhibit the in vitro fertilization of mice, while on human sperm This protein is not expressed. The present invention uses dequalinium chloride as a birth control drug for rodents, which can effectively control the combination of sperm and eggs of rodents, achieve control of the population of rodents, and achieve the purpose of controlling rodent damage without pesticides, and its natural enemies Taking this medicine is not harmful.

Description

Application of Dequalinium Chloride as Birth Control Drug in Mice

technical field

The invention relates to a birth control drug for rodents, in particular to the application of dequalinium chloride as a birth control drug for rodents.

Background technique

Dequalinium chloride (Dequalinium chloride, CAS#522-51-0), used as a PKC inhibitor as an anticancer drug, its IC50 is 7-18μM, and it is also a selective melittin-sensitive Potassium channel blocker with an IC50 of 1.1 μM ^{[1, 2, 3, 4]} . Recently, it was found that dequalinium chloride can also inhibit the activity of microbial-derived chitin hydrolase at the nanomolar level [5]. Dequalinium chloride has little toxicity to the human body, does not affect human reproduction, and is irritating to the eyes and skin. The lethal dose for mice is: 150-1000 mg/kg body weight orally, 70 mg/kg body weight for subcutaneous injection, and 1.9 mg/kg body weight for intravenous injection; the lethal dose for rabbits and dogs is 500 mg/kg body weight (MSDS).

Di-N-acetylchitobiase, or Di-N-acetylchitobiase (abbreviated as CTBS), is a kind of chitin hydrolase, and in mammals, it is only expressed in the liver of rodents and humans ^{[6, 7,8]} .

Reproductive isolation of species is a common natural phenomenon, and the interaction between carbohydrates on the zona pellucida of eggs and proteins on sperm is one of the mechanisms. It has been reported that Lewis oligosaccharide X (ie Lewis X) can bind to mouse sperm and can specifically inhibit the in vitro fertilization of mice, suggesting that the protein that recognizes Lewis X on the sperm may be involved in the reproductive isolation of mice ^{[9 ]} , but that's just a guess.

Rodent infestation refers to the harm caused by rodents to agricultural and other production. Rodents belong to Mammalia (Mam-malia) Rodentia (Rodentia) animals, a total of more than 1600 species. Rodents have many reproductive times, short gestation period, high litter rate, rapid sexual maturity, and the number can increase sharply in a short period of time; it has strong adaptability. It can survive in plains, mountains, forests, grasslands and even desert areas, often causing huge disasters to agricultural and other production.

Therefore, there is an urgent need to provide a medicine that can control the birth of rodents, especially a medicine that inhibits the combination of sperm and eggs of rodents, and the medicine can suppress rodent damage in the wild and its natural enemies are harmless to eat, and there is no need to use pesticides to suppress rodent damage.

References of the present invention are as follows:

Manetta A, et al. Gynecol Oncol, 1993, 50(1), 38-44.

Castle NA, et al. Eur J Pharmacol, 1993, 236(2), 201-207.

Dunn PM, et al. Eur J Pharmacol, 1994, 252(2), 189-194.

Rotenberg SA, et al. J Biol Chem, 1998, 273(4), 2390-2395.

Pantoom S, et al, J. Biol. Chem. 2011, 286:24312-24323.

Aronson NN Jr, et al, Arch of Biochem Biophy, 1989, 272(2), 290–300.

Liu B, et al, Glycobiology, 1999, 9(6), 589-593.

Park C, et al, J Biol Chem 2005, 280, 37204-37216.

Kerr CL, et al, Biol Reprod, 2004, 71(3), 770-777.

Rao FV, et al, Chem Bio, 2005, 12, 973–980.

Shur BD, et al, J Biol Chem 1988, 263, 17706–17714.

Contents of the invention

The purpose of the present invention is to provide a kind of application of the medicine that can control rodent fertility, especially provide a kind of application that can suppress the application of the medicine dequalinium chloride of the sperm-egg binding of rodent, this medicine can be used for controlling the population quantity of rodent , so as to achieve the effect of controlling rodent damage without using pesticides, and the natural enemies of rodents eat the drug without harm.

In order to achieve the above object, the present invention adopts the following technical solutions:

The application of dequalinium chloride as a birth control drug for rodents is used to control the population of rodents, so as to achieve the purpose of controlling rodent damage.

Further, the birth control of the rodents is to use the inhibitor dequalinium chloride to inhibit the sperm-egg combination of the rodents.

Further, the receptor protein specifically expressed on the sperm in the sperm-egg combination of the mouse is di-N-acetylchitobiase, namely Di-N-acetylchitobiase, abbreviated as CTBS; Quinium chloride is the receptor protein CTBS inhibitor specifically expressed on the sperm in the sperm-egg combination of described mice, and CTBS is an indispensable protein in the sperm-egg combination of mice, so dequalinium chloride is used as a mouse Class-specific birth control drug use.

We have found through research that CTBS is a unique sperm-egg binding receptor on rodent sperm, and its inhibitor dequalinium chloride can effectively inhibit in vitro fertilization in mice, and has little toxic effect on humans.

We found that CTBS is only expressed in rodent sperm, and CTBS antibody can inhibit in vitro fertilization in mice, but this protein is not expressed in human sperm.

The binding affinity of inhibitor dequalinium chloride of the present invention and CTBS is strong, and the activity of suppressing the combination of sperm and egg of mice is high, and experiment shows that it suppresses the semi-inhibition constant＜100nM of sperm-ovum combination; Further, its semi-inhibition constant is 50nM, therefore, dequalinium chloride can be used as a birth control drug for mice, and dequalinium chloride can inhibit the in vitro fertilization of mice, so dequalinium chloride can be used to control the population of mice, so as to achieve control The purpose of rodent infestation.

The present invention uses dequalinium chloride as a birth control drug for rodents, which can effectively control the combination of sperm and eggs of rodents, achieve control of the population of rodents, and achieve the purpose of controlling rodent damage without pesticides, and its natural enemies Taking this medicine is not harmful.

Description of drawings

Figure 1 is a graph showing the inhibition of dequalinium chloride on in vitro fertilization of mice.

Figure 2A is a schematic diagram of the identification of Lewis X receptors.

Figure 2B is a map of mouse sperm proteins found using Lewis X probes.

Figure 2C is the mass spectrometry analysis profile of the protein.

Figure 3 is a picture of CTBS antibody inhibiting the binding of Lewis X to mouse sperm.

Figure 4 is a picture of the binding position of Lewis X on mouse sperm and the localization results of CTBS on mouse sperm.

Fig. 5A is a schematic diagram of the interaction between the recombinant human CTBS and the mouse egg zona pellucida.

Figure 5B is the bright field and fluorescence photographs of the interaction between the recombinant human CTBS and the mouse egg zona pellucida.

Figure 5C is a western blot analysis of RFP or RFP-CTBS enriched in latex particles with CTBS antibody.

Fig. 6 is a graph showing the induction effect of CTBS antibody on mouse sperm acrosome reaction.

Fig. 7 is a diagram showing that different concentrations of CTBS antibodies inhibit the in vitro fertilization of mice.

Figure 8 is a diagram of Western blot analysis of mouse, rat and human sperm lysates.

Detailed ways

The implementation of the present invention will be illustrated by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

Embodiment 1: the application of dequalinium chloride as the birth control drug of mice

Each dosage experiment of different concentrations of CTBS uses >50 mouse eggs, repeats 3 times, and each dosage uses >150 eggs in total. The experimental results are shown in Figure 1. Ammonium can well inhibit in vitro fertilization in mice.

Example 2:

We found that CTBS is expressed in rodent sperm, and CTBS antibody can inhibit in vitro fertilization in mice, but this protein is not expressed in human sperm. Its experimental method is as follows:

(1) Discovery of CTBS on mouse spermatozoa:

CTBS on mouse sperm was identified by using Lewis X (Lewis X, abbreviated as LeX) probe, and it was proved that CTBS can bind to mouse egg zona pellucida.

(1) First identify the Lewis X receptor. The identification process is shown in Figure 2A, including the identification process of the lectin-like protein that can recognize Lewis X and the identification process of the endogenous protein containing Lewis X modification. The principle is to modify the surface of bovine serum albumin (BSA) with Lewis X to make the pseudoglycoprotein LeX-BSA, and further modify the pseudoglycoprotein with ultraviolet crosslinking reagent [6-(biotinamido)-2-( p-Azidobenzamido)caproylamino]ethyl-1,3'-dithiopropionic acid sulfosuccinimide ester (Sulfo-NHS-2-(6-[biotinamido]-2-( p-azido benzamido)-hexanoamido)ethyl-1,3'-dithiopropionate (Sulfo-SBED for short) was used as LewisX probe. The probe is cross-linked to mouse sperm by ultraviolet cross-linking to form a Lewis X probe-receptor complex, and the lotus tetragonolobus agglutinin (LTA) resin that can recognize Lewis X is used to separate and purify the sperm cell lysate The Lewis X probe-receptor complex in . Since mouse sperm may contain endogenous Lewis X-modified proteins, an endogenous Lewis X-modified protein identification process was set up as a control to distinguish such proteins (control group). Generally speaking, the differential proteins identified by the two processes may be the receptor protein of Lewis X.

(2) Proteins on mouse sperm found using Lewis X probe:

Use the Lewis X probe to find the mouse sperm protein, as shown in Figure 2B. The differential bands 1, 2, 3, and 4 in Figure 2B were removed for mass spectrometry identification of the protein, as shown in the mass spectrometry analysis chart in Figure 2C, after analysis, the identities of proteins that may interact with carbohydrates are shown in Table 1 shown.

Table 1

NOTE: The first column in Table 1 is the nucleoplasmic ratio, the second column is the peptide sequence, and the third column is the protein identity.

(3) The CTBS antibody can inhibit the binding of Lewis X fluorescent probe to mouse sperm:

Lewis X fluorescent probe LeX-BSA-Alexa430 is prepared from the LewisX pseudoglycoprotein LeX-BSA plus fluorescent group Alexa430. As shown in Figure 3, the probe can bind to mature spermatozoa extracted from the tail of the mouse epididymis (positive control). The antibody is a rabbit polyclonal antibody prepared from human CTBS recombinant protein, purchased from Wuhan Sanying Biotechnology Co., Ltd.

After research, it was found that only CTBS antibody could inhibit the binding of LeX-BSA-Alexa430 to mouse sperm, as shown in Figure 3. Moreover, a weak chitinase inhibitor pentoxifylline (PTX) can also inhibit this binding [10], excluding the non-specific binding inhibition caused by the steric hindrance effect of the antibody itself.

(4) The binding position of Lewis X on mouse sperm is consistent with the location of CTBS on mouse sperm:

Lewis X fluorescent probe LeX-BSA-Alexa430 is prepared from the LewisX pseudoglycoprotein LeX-BSA plus fluorescent group Alexa430. The probe can bind to mature sperm taken from the tail of the mouse epididymis, as shown in the inset of Figure 4, and the binding situation is consistent with previous reports [9]. The antibody is a rabbit polyclonal antibody prepared from human CTBS recombinant protein, purchased from Wuhan Sanying Biotechnology Co., Ltd. Through immunofluorescence localization, we found that the binding position of LeX-BSA-Alexa430 on mouse sperm was consistent with the CTBS protein localization of mouse sperm, as shown in Figure 4.

(5) Interaction between recombinant human CTBS (i.e. rCTBS) and mouse egg zona pellucida (ZP):

A. The experimental idea is shown in Figure 5A. Red fluorescent protein (RFP) antibody was immobilized on the latex particles, and the recombinant RFP-CTBS protein expressed in 293 cells was enriched on the latex particles, and added to the medium containing mouse eggs. medium. If CTBS can recognize carbohydrates on the zona pellucida of mouse eggs, latex particles with red fluorescence will be attached to the zona pellucida of mice.

B. Figure 5B is a bright field (left) and red fluorescence (right) photo. It was found that latex particles enriched only to RFP could not bind to the zona pellucida of mouse eggs (as shown in the upper row of Figure 5B); while latex particles with RFP-CTBS could bind to the zona pellucida of mouse eggs ( As shown in the middle row of Figure 5B), while CTBS antibody can block this binding (as shown in the lower row of Figure 5B). The fluorescence photographs on the right confirm the above observations.

C. Figure 5C is the result of Western blot analysis, and the graphical result shows that latex particles bound with RFP antibody can enrich RFP or RFP-CTBS. The white arrows refer to RFP, and the black arrows refer to RFP-CTBS.

The above results prove that the recombinant human CTBS can interact with the mouse egg zona pellucida.

(B) CTBS is involved in the fertilization process in mice.

(1) Induction effect of CTBS antibody on mouse sperm acrosome reaction:

The CTBS antibody of different concentrations between 0-30ug/ml is used to induce the acrosome reaction of mouse sperm, as shown in Figure 6, the results prove that CTBS antibody can induce the acrosome reaction of mouse sperm, which is different from the small reported in the prior art. The characteristics of receptor proteins related to sperm-egg binding on mouse sperm are consistent ^[11] .

(2) Different concentrations of CTBS antibody inhibited in vitro fertilization of mice:

Different concentrations of CTBS antibodies were used to inhibit the in vitro fertilization of mice, as shown in Figure 7, the results proved that CTBS antibodies can well inhibit the in vitro fertilization of mice, further indicating that CTBS on mouse sperm is indeed combined with mouse sperm eggs related.

(3) Western blot analysis of mouse, rat and human sperm lysate.

Western blot analysis was performed on sperm lysates from mice, rats and humans. The sample volume of sperm lysates was 50ug for mice, 60ug for rats, and 60ug for humans. The antibody is a rabbit polyclonal antibody prepared from human-derived CTBS recombinant protein, which was purchased from Wuhan Sanying Biotechnology Co., Ltd.; the sample volume of the human cell line Hela cell lysate was 10ug; alpha-tubulin (α-tubulin ) as a reference for protein loading. The results are shown in Figure 8, indicating that the CTBS antibody has high sensitivity to human-derived CTBS, which further proves that CTBS is not expressed on human sperm, but only expressed on rodent sperm.

In summary, all the results described in Example 1 and Example 2 show that CTBS is a receptor protein specifically expressed on rodent sperm, which participates in the physiological process of sperm-egg binding, and the CTBS antibody can inhibit the mouse in vitro Fertilize. As a birth control drug for mice, dequalinium chloride can well inhibit the in vitro fertilization of mice, and experiments have shown that its half-inhibition constant is <100nM.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

Claims (3)

1. The application of dequalinium chloride as a birth control drug for mice. 2. the application of dequalinium chloride as claimed in claim 1 as the birth control drug of rodents is characterized in that, described dequalinium chloride controls the fertility of rodents by inhibiting the combination of sperm and eggs of rodents. 3. the application of dequalinium chloride as claimed in claim 2 as the birth control drug of rodents, is characterized in that, described dequalinium chloride inhibits the receptor protein CTBS of specific expression on the sperm to suppress described mouse Kind of sperm-egg union.