阅读说明：本技术 一种抑制蚊子生殖的试剂及其应用 (Reagent for inhibiting mosquito reproduction and application thereof ) 是由 沈波 厉茜茜 张�成 孙艳 周丹 马磊 朱昌亮 于 2021-05-11 设计创作，主要内容包括：一种抑制蚊子生殖的试剂及其应用,属于蚊媒病防治技术领域,尤其涉及MG132在制备抑制蚊子生殖试剂中的应用。本发明利用MG132能够显著抑制蚊的生殖力,且这种抑制作用能够持续到子二代。(A reagent for inhibiting mosquito reproduction and an application thereof belong to the technical field of mosquito-borne disease control, and particularly relate to an application of MG132 in preparation of a reagent for inhibiting mosquito reproduction. The invention can obviously inhibit the fertility of mosquitoes by using MG132, and the inhibition effect can last to the second generation.)

Use of MG132 in the preparation of an agent for inhibiting mosquito reproduction.

2. The use of claim 1, wherein the working concentration of MG132 is 4 μ Μ.

3. An agent for inhibiting mosquito reproduction, characterized in that the active ingredient contains MG 132.

Technical Field

The invention belongs to the technical field of mosquito-borne disease control, and particularly relates to a reagent for inhibiting mosquito reproduction and application thereof.

Background

The mosquito is a kind of pest, and has the habit of blood sucking, and when the blood is sucked, the mosquito can inject the saliva of plasmodium, cercaria and encephalitis B virus into human body to infect human body. Therefore, mosquitoes not only suck blood to affect sleep but also transmit encephalitis B, malaria, dengue fever, filariasis and the like. At present, mosquito vectors are still the main means for controlling mosquito vectors, and the main mosquito-proof means are divided into the following: 1. the environment is controlled, and the purpose is to clear away the places where mosquitoes and insects propagate. The treatment method can be divided into a. environmental modification: land, water or vegetation is modified to prevent, eliminate and reduce mosquito breeding and inhabitation. Comprises the measures of removing accumulated water, filling soil and blocking holes, eradicating weeds and the like. b. And (3) environmental treatment: the aquatic environment is changed in a planned way, so that the mosquito breeding is not facilitated. Dredging the ditch, changing still water into running water and the like. c. Reducing human mosquito contact: the contact between human beings and mosquitoes is reduced by improving the living conditions and habits of people. The house is provided with a screen door and a screen window, a mosquito net is used, and the mosquito net is treated by medicaments. The mosquito-proof cap or the insect-repelling net can be worn for outdoor duty operation. 2. The chemical control has the advantages of quick effect, convenient use, suitability for large-scale application and the like, is one of important means for mosquito control at present, and is mainly divided into a larva control: the organophosphorus pesticide and its slow releasing agent may be added into water, and the commonly used pesticide includes fenthion and fenthion. b. Adult mosquito control: (1) space spraying: insecticidal aerosols are used to kill flying and perching insects. Such as using aerosol cans, ultra low volume sprays, hot aerosols, and the like. (2) Retention spraying: the insecticide with a certain lasting effect is sprayed on the surface where mosquitoes inhabit indoors, and the used insecticide comprises high-efficiency cypermethrin, deltamethrin, cyhalothrin and the like. 3. Biological control: high cost and difficult large-scale implementation. Because the long-term mass use of the chemical insecticide causes the occurrence and development of mosquito insecticide resistance, the novel mosquito population inhibition method based on the direct inhibition of the reproductive potential energy of female mosquitoes has more important practical application value for controlling the mosquito population quantity.

MG132 (Proteasome inhibitor) is a potent, reversible, cell-permeable Proteasome inhibitor (Ki =4 nM). N-benzyloxycarbonyl-L-leucyl-L-leucine MG132 belongs to a synthetic peptide aldehyde inhibitor. It reduces the degradation of ubiquitin-conjugated proteins by 26S complexes in mammalian cells and yeast permeable strains without affecting their atpase or peptidase activities. MG132 activates c-Jun N-terminal kinase (JNK 1), which initiates apoptosis.

MG132 also inhibits NF-. kappa.B activation with an IC 50 of 3. mu.M, and blocks β -secretase activity. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC 50 of about 50 μ MMG132 for 24 hours. MG132 inhibits the growth of HeLa cells by inducing cell cycle arrest and triggering apoptosis. MG132 inhibited C6 glioma cell proliferation in a time and dose dependent manner (IC 50 value of 18.5 μ M at 24 hours). MG132 (18.5 μ M) inhibited proteasome activity by about 70% at 3 hours.

MG132 induces apoptosis by down-regulating the anti-apoptotic proteins Bcl-2 and XIAP, up-regulation of the pro-apoptotic proteins Bax and caspase-3, and production of cleaved C-terminal 85kDa PARP. MG132 also increases active oxygen by more than 5-fold. The effect of IC 50MG132 on cell viability of HeLa, CaSki and C33A cervical cancer cells after 48 hours of incubation was 2.1,3.2 and 5.2 μ M, respectively. The in vivo antitumor activity of MG132 against cervical cancer was examined using sc xenograft model.

MG132 was injected at 1MG/kg using the following protocol: for HeLa tumor bearing mice, days 1,4,8,12,15,18,23, and 26. The growth inhibition rate of MG132 was 49% compared to the control. MG132 (ip, 0.1 MG/kg/day) reduces cardiac hypertrophy caused by pressure overload and improves cardiac function of Abdominal Aorta Banding (AAB) rats by regulating ERK1/2 and JNK1 signal pathways.

Disclosure of Invention

The technical problem to be solved is as follows: the invention aims at the technical problem and provides a reagent for inhibiting the reproduction of mosquitoes and application thereof.

The technical scheme is as follows: use of MG132 in the preparation of an agent for inhibiting mosquito reproduction.

Preferably, the MG132 is used at an operating concentration of 4 μ M.

An agent for inhibiting mosquito reproduction contains MG132 as effective component.

Has the advantages that: the invention can obviously inhibit the fertility of mosquitoes by using MG132, and the inhibition effect can last to the second generation.

Drawings

FIG. 1: effect of MG132 on reproductive action of parental female mosquitoes. A: the effect of MG132 on the amount of eggs laid by the parental female mosquitoes; b: effect of MG132 on parent female mosquito larvae count; c: effect of MG132 on egg hatchability (. sup.)P<0.05，**P<0.01，***P<0.0001）。

FIG. 2: effect of MG132 on reproductive action of female mosquitoes of a next generation. A: the effect of MG132 on the oviposition of the next generation of female mosquitoes; b: the effect of MG132 on the number of offspring female mosquitoes; c: effect of MG132 on egg hatchability (N =20, etc.)P<0.05，**P<0.01，***P<0.0001）。

FIG. 3: the effect of MG132 on reproduction of the next generation female mosquitoes. A: the effect of MG132 on the oviposition of the next generation female mosquito. B: the effect of MG132 on the number of larvae of the second generation female mosquitoes. C: effect of MG132 on egg hatchability (N =20, etc.)P<0.05，**P<0.01，***P<0.0001）。

FIG. 4: the effect of MG132 on the reproductive activity of the third generation female mosquitoes. A: the effect of MG132 on the oviposition of the third generation female mosquitoes. B: the influence of MG132 on the number of the female mosquito larvae of the third generation; c: effect of MG132 on egg hatchability (N =20, etc.)P<0.05，**P<0.01，***P<0.0001）。

FIG. 5: parental MG132 treatment groups had an anopheles dissected view of the blood-sucking, non-oviposition female mosquito. A: a 72 h PBM ovary dissection map of a normal female mosquito; B-G: parental MG132 treatment group blood-sucking 5 d non-oviposition female mosquito ovary anatomical map (magnification 3.2x 10).

FIG. 6: MG132 treatment group parental and offspring female mosquito ovary morphology ratio.

Detailed Description

The following examples are presented to enable one of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way.

Example 1

Materials and methods

Effect of MG132 on mosquito fecundity

MG132 mother liquor preparation

47.5 MG of MG132 (Synonyms: Z-Leu-Leu-Leu-al; MG132) powder (MCE, HY-13259) was weighed and dissolved in 1 mL of DMSO (MCE, HY-129770) to mix well and prepare 100 mM MG132 mother liquor.

2. Determination of the Effect of MG132 on mosquito fecundity

Taking 6 mu L of the MG132 mother liquor, adding 150 mL ddH₂O, preparing 4 mu M working solution. Treating 3-year-old late-to-4-year-old primary larvae with the working solution until eclosion is carried out for 3 days, and treating with DMSO and ddH with equal concentration₂O treatment served as a control. Each group treated 500 larvae and 3 biological replicates were set up.

After 3 days of eclosion, male and female mosquitoes are fully mated, blood is fed, and the egg laying amount of each group of female mosquitoes is observed 72 h (72 h PBM) after blood suction. The number of eggs in each raft was counted under an OLMPUS phase contrast inverted microscope (N = 20), the rafts were returned to the laboratory for normal rearing, the larvae were allowed to hatch, 1 instar larvae were counted, and hatchability was calculated (N = 20).

In addition, the ovaries of female mosquitoes 72 h PBM female mosquitoes were dissected and the change in the morphology of the ovaries of each group was observed under an OLMPUS phase contrast inverted microscope.

Results

Effect of MG132 on mosquito fecundity

Changes in the amount of eggs laid, number of larvae and hatchability of female mosquitoes after MG132 treatment

In the parents, the egg laying amount of mosquitoes in a DMSO control group is 140.1 +/-9.42, the number of larvae is 119 +/-11.64, the hatching rate is 84.91% +/-4.85%, while the egg laying amount of mosquitoes in an MG132 treatment group is only 81.25 +/-46.28, the number of larvae is 65.29 +/-38.27, the hatching rate is 63.48% +/-34.85%, the egg laying amount of the MG132 treatment group is obviously lower than that of the control group (figure 1,P<0.0001、 P<0.0001、 P= 0.0016). To study the persistence of this inhibition, we subsequently observed the treatment of mosquitoes to three generations consecutively, and found that there was no significant difference in the egg laying amount, larva number and hatchability of the offspring of the DMSO control group from the parent of the control group, while the egg laying amount, larva number and hatchability of the offspring after MG132 treatment were 104.48 ± 42.58, 87.52 ± 27.62 and 73.06% ± 27.62%, respectively; the egg laying amount, the number of larvae and the hatching rate of the second generation treated by MG132 are respectively107.72 + -33.49, 97.32 + -32.07, 86.31 + -17.95%; the egg laying amount, the number of larvae and the hatchability of the third generation treated by MG132 are 166.95 +/-20.95, 146.75 +/-21.41 and 87.66 +/-4.00 respectively. Suggesting that the egg laying amount, the number of larvae and the hatching rate of the first filial generation after the MG132 treatment are remarkably reduced (figure 1,P<0.0001， P<0.0001， P=0.0048）。

the egg laying amount and the number of larvae of the second generation treated by MG132 are obviously reduced, and the hatchability is not different (figure 3,P<0.0001， P<0.0001，P=0.2267）。

the oviposition amount, the number of larvae and the hatchability index of the third generation treated by MG132 have no obvious difference (figure 4,P<0.0001，P<0.0001，P= 0.0048). After the MG132 inhibits the proteasome activity, the reproductive capacity of the mosquitoes can be obviously inhibited, and the inhibition effect can be continued to the second generation. In the parental MG 132-treated group and the offspring, 6 and 3 female mosquitoes that suck blood for 5 days were observed to have not spawned yet, respectively. The parental female mosquito ovaries were dissected and 1 ovary was found to have significantly reduced volume, 5 ovaries had delayed development and the follicles had not developed (fig. 5). Suggesting that the effect on mosquito fertility after MG132 treatment may be achieved by affecting ovarian development.

We further observed changes in ovarian morphology following MG132 treatment, parental, first-, second-, and third-generation MG 132-treated groups and control groups, each group randomly collected 20 female mosquito ovaries of 72 h PBM for dissection. The ovaries were divided into four groups according to their different morphology: normal morphology group, sparse morphology group, volume-variant group and delayed development group. The results show that the ovary morphology normal proportion of parental generation, first generation and second generation is 10%, 15.8% and 50%, respectively, and the ovary morphology normal proportion of the female mosquito of the MG132 treatment group is gradually increased along with the reduction of the MG132 action. After treatment, the ovarian morphology of the third generation had recovered to normal (fig. 6).