CN111358936A

CN111358936A - Application of NRF2 protein in preparing medicine for regulating biological rhythm

Info

Publication number: CN111358936A
Application number: CN202010200520.2A
Authority: CN
Inventors: 陈海龙; 曲丽娜; 李莹辉; 吕柯; 费俊龙; 冀国华; 元艳宏; 卢亮; 梁峰吉; 李铠
Original assignee: 63919 Troops of PLA
Current assignee: 63919 Troops of PLA
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-07-03

Abstract

The invention discloses an application of NRF2 protein in preparing a medicament for regulating biological rhythm, wherein the NRF2 agonist is bortezomib. The invention has the beneficial effects that: can realize the quick adjustment and the interference-free adjustment of the biological rhythm, and specifically comprises the following steps: 1) by directly activating the NRF2 protein, the NRF2 quickly enters the nucleus to play a role, and a slow regulation way of 'increasing the NRF2 content' in the process of 'DNA-mRNA-protein' is avoided; 2) the combination of NRF2 protein and genes related to biological rhythm is promoted, so that the content of biological rhythm protein is directly increased, the biological rhythm is rapidly adjusted, and a slow adjusting way of indirectly increasing the content of the biological rhythm protein through biological rhythm feedback protein is avoided; and avoids the interference of the biorhythm feedback protein on the biorhythm protein content.

Description

Application of NRF2 protein in preparing medicine for regulating biological rhythm

Technical Field

The invention relates to the technical field of biological rhythm regulation, in particular to application of NRF2 protein in preparation of a medicament for regulating biological rhythm.

Background

Biorhythms are life activities that appear at various levels in the body, such as molecules, cells, organs, systems, and behaviors, in a time sequence and in cycles. Biorhythms widely regulate and control various physiological functions such as body temperature, cardiovascular activity, respiration, immune response, hormone secretion, behavior, cognitive function, and the like.

The core mechanism of biorhythms is the transcription of biorhythmic genes and the molecular oscillation formed by post-transcriptional regulation. The CLOCK and BMAL1 proteins are molecular markers and cores of biological rhythms. After the rhythm gene is started, the rhythm gene is transcribed and translated to generate corresponding protein, and when the protein concentration reaches a certain degree, the protein is fed back to act on the gene promoter, so that the concentration of the protein is in 24h periodic oscillation.

The self-oscillation of rhythmic gene transcription consists of multiple feedback loops, of which 2 are most important, as shown in fig. 1:

(1) CLOCK and BMAL1 protein form heterodimer, bind to rhythm genes PER1-3 and CRY1/2, and activate gene transcription thereof. The PER and CRY series proteins are transferred from cytoplasm to nucleus and act with CLOCK and/or BMAL1 to inhibit the activity of CLOCK-BMAL1, thereby inhibiting the transcription of PER1-3 and CRY 1/2.

(2) CLOCK-BMAL1 also activates transcription of orphan nuclear receptor REV-ERB α, and its expression protein REV-ERB α can bind with Bmal1 gene to repress transcription of BMAL 1.

The gene transcription and the protein entering the nucleus need a certain time, so that the period of the oscillation of the rhythm molecules is just maintained at about 24 h. This molecular oscillation not only expressed the PER1-3 and CRY1/2 in the biological rhythm, but also provided the transcriptional activity of the CLOCK-BMAL1 heterodimer with a rhythmic character.

The NRF2 protein can indirectly regulate the expression of CLOCK-BMAL1 in the body. The NRF2 protein is a major regulator of cellular oxidative stress. Normally, NRF2 is present in the cytoplasm of cells and is maintained at a low level. Wherein, the combination of Kelch-like ECH-associated protein 1(KEAP1) and NRF2 induces the degradation thereof, so that NRF2 is in an inactivated state. Under oxidative stress conditions, KEAP1 oxidizes, causing it to dissociate from NRF2, releasing NRF2 from degradation, allowing NRF2 to accumulate in the cytoplasm and transfer into the nucleus. With the aid of other coactivating proteins, NRF2 binds to phase II and antioxidant enzyme genes, initiating expression of these enzyme genes, ultimately reducing intracellular ROS levels to ameliorate oxidative damage such as lipid peroxidation. In addition to promoting the dissociation of NRF2 and KEAP1, oxidative stress can induce the phosphorylation of NRF2 by various kinases such as PKC, MAPK, PI3K/AKT, CK2, etc., leading to their nuclear entry and activation, as shown in fig. 2. NRF2 was able to induce expression of more than 250 genes.

Research shows that the REV-ERB α gene as an important link in the feedback loop of CLOCK-BMAL1 has NRF2 regulating sequence, so far, NRF2 has been found to indirectly regulate the content of CLOCK and BMAL1 by influencing REV-ERB α and CRY 2.

While NRF2 protein indirectly regulates biorhythms: the NRF2 protein is combined with genes Rev, ROR, Per and Cry corresponding to biorhythm feedback factors REV, ROR, PER and CRY, etc., to change the expression of REV, ROR, PER and CRY in cell nucleus, thereby changing their content. After the factors go out of the nucleus, the genes CLOCK and Bmal1 corresponding to the rhythm proteins CLOCK and BMAL1 are combined in a related way, so that the expression of the rhythm proteins CLOCK and BMAL1 in the nucleus is changed, the content of the CLOCK and BMAL1 proteins is changed, and the biological rhythm is further changed, as shown in figure 3.

However, the prior art also has the defects of slow response speed and easy interference of the biological rhythm regulation technology.

The prior art changes the content of the rhythm protein by increasing the content of NRF2 protein to ensure that the NRF2 protein is combined with genes of biological rhythm related feedback factors REV and ROR to change the content of the REV and ROR, and then the factors enter a cell nucleus to be combined with the gene of the rhythm gene Bmal 1. This technique involves 3 steps:

(1) increasing NRF2 protein content;

(2) NRF2 binds to genes of biorhythm feedback factors REV, ROR, etc., thereby changing the contents of REV, ROR;

(3) REV, ROR alter the content of rhythmic proteins.

The steps relate to the processes of combining a plurality of proteins with genes, expressing the genes into the proteins and the like, and the reaction speed is low; the steps (2) and (3) of the content change of REV and ROR and the content change of rhythm protein of REV and ROR have a plurality of compensation modes in organisms. For example, despite changes in REV, ROR, it is possible that other feedback factors such as CRY, etc. compensate quickly, resulting in an unexpected change in the content of rhythmic proteins and thus are susceptible to interference.

Disclosure of Invention

The invention aims to overcome the defects of slow response, easy interference and the like of the biorhythm regulation technology in the prior art, and provides the application of the NRF2 protein in preparing a medicament for regulating biorhythm. Can effectively improve the regulation speed of the biological rhythm, reduce the interference of other factors in the cell to the regulation of the biological rhythm and realize the quick regulation and the interference-free regulation of the biological rhythm.

In order to achieve the purpose, the technical scheme provided by the invention is as follows: use of NRF2 protein in the manufacture of a medicament for modulating a biological rhythm.

Further, in the application, the amino acid sequence of the NRF2 protein is shown as a sequence 1 in a sequence table.

The sequence is abbreviated as:

MDLIDILWRQDIDLGVSREVFDFSQRRKEYELEKQKKLEKERQEQLQKEQEKAFFTQLQLDEETGEFLPIQPAQH TQSETSGSANYSQVAHIPKSDALYFDDCMQLLAQTFPFVDDNEVSSATFQSLVPDIPGHIESPVFIATNQAQSPETSVA QVAPVDLDGMQQDIEQVWEELLSIPELQCLNIENDKLVETTMVPSPEAKLTEVDNYHFYSSIPSMEKEVGNCSPHFLNA FEDSFSSILSTEDPNQLTVNSLNSDATVNTDFGDEFYSAFIAEPSISNSMPSPATLSHSLSELLNGPIDVSDLSLCKAF NQNHPESTAEFNDSDSGISLNTSPSVASPEHSVESSSYGDTLLGLSDSEVEELDSAPGSVKQNGPKTPVHSSGDMVQPL SPSQGQSTHVHDAQCENTPEKELPVSPGHRKTPFTKDKHSSRLEAHLTRDELRAKALHIPFPVEKIINLPVVDFNEMMS KEQFNEAQLALIRDIRRRGKNKVAAQNCRKRKLENIVELEQDLDHLKDEKEKLLKEKGENDKSLHLLKKQLSTLYLEVF SMLRDEDGKPYSPSEYSLQQTRDGNVFLVPKSKKPDVKKN。

further, the above application, the regulation of biorhythm is realized by the following two ways:

1) modulation of biological rhythms is achieved by activation of NRF2 protein;

2) the regulation of biological rhythm is realized by promoting the combination of NRF2 protein and genes related to biological rhythm.

Further, in the above application, the activation of the NRF2 protein and the promotion of the binding of the NRF2 protein to the genes related to the biological rhythm are both realized by the NRF2 agonist.

Further, for the above use, the NRF2 agonist is bortezomib.

The invention has the beneficial effects that: the NRF2 protein provided by the invention can be applied to the preparation of the medicine for regulating the biological rhythm, can realize the quick regulation and the interference-free regulation of the biological rhythm, and specifically comprises the following steps:

quick adjustment:

one is that the NRF2 can rapidly enter the nucleus to play a role by directly activating the NRF2 protein. Avoids the slow regulation pathway of the DNA-mRNA-protein process of increasing the NRF2 content.

And secondly, the NRF2 protein is promoted to be combined with genes related to the biological rhythm, so that the content of the biological rhythm protein is directly increased, and the biological rhythm is rapidly regulated. Avoids the slow regulation way of indirectly increasing the content of the biorhythm protein through the biorhythm feedback protein.

(II) non-interference regulation:

the combination of NRF2 protein and genes related to biological rhythm is promoted, so that the content of biological rhythm protein is directly increased, and the interference of biological rhythm feedback protein on the content of biological rhythm protein is avoided.

Drawings

FIG. 1 shows a biorhythmic gene transcription feedback loop for mammals.

FIG. 2 shows that NRF2 activates and promotes downstream antioxidant enzyme gene expression under stress conditions.

FIG. 3 shows that NRF2 protein indirectly regulates biological rhythm.

Figure 4 shows the effect of different NRF2 siRNA 100nM treatments for 24h on CLOCK, BMAL1 expression in NIH/3T3 cells, where the data are expressed as mean ± standard deviation and n is 4. P <0.05 compared to NC-FAM treatment group.

Figure 5 shows the effect of NRF2 siRNA-1208 treatment at different concentrations for 24h on CLOCK, BMAL1 expression in NIH/3T3 cells, where the data are expressed as mean ± standard deviation and n is 4. P <0.05, p <0.01 compared to NC-FAM treatment group.

Figure 6 shows the effect of different concentrations of Brusatol treatment for 4h on NIH/3T3 cell viability, where data are presented as mean ± standard deviation, n-4.

Figure 7 shows the effect of different concentrations of Brusatol treatment for 4h on NIH/3T3 cell CLOCK, BMAL1 protein expression, where data are expressed as mean ± standard deviation, n ═ 4, × <0.05, × <0.01 compared to DMSO treatment group.

Figure 8 shows the effect of pCMV6-Entry-myc-nrf2 plasmid treatment for 24h on expression of CLOCK, BMAL1 in NIH/3T3 cells, where the data are expressed as mean ± standard deviation, n is 4, p <0.05 compared to 2000ng NC-myc treated group.

Figure 9 shows the effect of NRF2 siRNA treatment for 24h on horse serum induction of the rhythmicity of NIH/3T3 cells CLOCK, BMAL1, where the data are expressed as mean ± standard deviation, n-4; HS, horse serum; p <0.05NRF2 siRNA + HS treated group compared to the time point corresponding to HS treated group.

Figure 10 shows the effect of brustol treatment for 4h on horse serum induction of rhythmic expression of NIH/3T3 cells CLOCK, BMAL1, where data are expressed as mean ± standard deviation, n-4; HS, horse serum; p <0.05 is the time point for the Brusatol + HS treatment group compared to the HS treatment group.

Figure 11 shows the effect of NRF2 agonist BTZ and inhibitor ATRA on NRF2 activity in NIH/3T3 cells, where the data are expressed as mean ± standard deviation, n-4; p <0.05, p < 0.01; A. effect of different concentrations of BTZ treatment for 24h on both cytoplasmic and nuclear NRF2 activity of NIH/3T3 cells; B. effects of ATRA and BTZ treatment for 24h on cytoplasmic and nuclear NRF2 activity of NIH/3T3 cells.

Figure 12 shows the effect of different concentrations of BTZ treatment for 24h on NIH/3T3 cell cytoplasm and nuclear CLOCK, BMAL1 expression, where data are expressed as mean ± standard deviation, n-4; p <0.05, p <0.01 compared to 0.01% DMSO.

Figure 13 shows the effect of BTZ and ATRA treatment for 24h on NIH/3T3 cell cytoplasm and nuclear CLOCK, BMAL1 expression, where data are expressed as mean ± standard deviation, n-4; p <0.05, p < 0.01.

Figure 14 shows the effect of BTZ and ATRA treatment for 24h on the rhythmicity of NIH/3T3 cells CLOCK, BMAL1, where data are expressed as mean ± standard deviation, n-4; p <0.05 compared to DMSO treated group.

Figure 15 shows the effect of BTZ and ATRA treatment for 24h on NIH/3T3 cells NRF2 binding to the Clock, Bmal1 gene, where data are expressed as mean ± standard deviation, n is 4; A. the effect of BTZ treatment for 24h on the binding of NRF2 to Clock and Bmal1 genes of NIH/3T3 cells; B. effects of BTZ and ATRA treatment for 24h on the binding of NRF2 to Clock, Bmal1 genes in NIH/3T3 cells.

Detailed Description

Example 1:

the regulation of biological rhythms by the NRF2 protein was studied for the purposes of: the research on the effect of NRF2 protein on biorhythm is carried out by taking a mouse embryo fibroblast cell line NIH/3T3 cell as a research object and researching the expression of NRF2 on biorhythm protein CLOCK and BMAL1 and the change of rhythmicity by reducing the synthesis of NRF2 protein, promoting the decomposition of NRF2 and increasing the synthesis of NRF2 respectively.

The procedure and results are shown below.

Firstly, the reduction of the synthesis of NRF2 can regulate the content of the rhythmic proteins CLOCK and BMAL 1:

1.NRF2 siRNA screening capable of reducing NRF2 synthesis:

a. the experimental method comprises the following steps:

RNA interference techniques are common techniques for reducing protein synthesis. The RNA interference agent siRNA is completely matched and combined with messenger RNA corresponding to target protein by a cell transfection technology, so that the transcription and translation of the target protein are inhibited, and the aim of reducing the synthesis of the target protein in cells is fulfilled.

The experimental groups were as follows (n ═ 4):

(1) DMEM group: culturing NIH/3T3 cells with a normal medium DMEM for 24 h;

(2) Opti-MEM group: culturing NIH/3T3 cells for 6h by using a cell transfection medium Opti-MEM, and then culturing for 18h by using a normal medium DMEM;

(3) lipo2000 group: NIH/3T3 cells were cultured in cell transfection medium Opti-MEM containing cell transfection reagent liposome 2000(Lipo2000) for 6h, and then in DMEM normal medium for 18 h;

(4) NC-FAM group: NIH/3T3 cells were transfected with green fluorescence control NC-FAM, i.e., cells were cultured in Opti-MEM medium containing cell transfection reagents Lipo2000 and NC-FAM for 6h, and then cultured in DMEM medium for 18 h;

(5) SiRNA-331 group: transfecting NIH/3T3 cells with an RNA interference agent SiRNA-331, namely culturing the cells in Opti-MEM (Opti-MEM) culture medium containing a cell transfection reagent Lipo2000 and the RNA interference agent SiRNA-331 with 100nM, and then culturing the cells in a normal medium DMEM for 18 h;

(6) SiRNA-793 group: transfecting NIH/3T3 cells with an RNA interference agent SiRNA-793, namely culturing the cells in Opti-MEM culture medium containing cell transfection reagent liposome 2000(Lipo2000) and the RNA interference agent SiRNA-793 with the cell transfection reagent of 100nM for 6h, and then culturing the cells in DMEM normal culture medium for 18 h;

(7) SiRNA-1208 group: NIH/3T3 cells were transfected with the RNA interference agent SiRNA-1208 by culturing the cells in Opti-MEM medium containing the cell transfection reagent liposome 2000(Lipo2000) and the RNA interference agent 100nM SiRNA-1208 for 6h, followed by culturing in DMEM medium, which is normal medium, for 18 h.

The sequence of NRF2 siRNA corresponding to each group is shown in the following table 1.

TABLE 1 NRF2 siRNA sequences

Then, each group of cells is collected, and the expression of biorhythm protein CLOCK, BMAL1, NRF2 protein and reference protein β -actin (used for indicating total protein content in the immunoblotting technology) in the cells is detected by the immunoblotting technology.

b. The experimental results are as follows:

compared with the NC-FAM treatment group, the NRF2 expression of the siRNA-1208 treatment group is remarkably reduced, and the NRF2 expression is not changed by the siRNA-331 or siRNA-793 treatment. siRNA-1208 is therefore able to reduce NRF2 synthesis.

Compared with the NC-FAM treated group, the expression of CLOCK and BMAL1 in the cells is remarkably reduced after the siRNA-1208 treatment (p is less than 0.05), and the figure 4 shows.

c. Experiment summary

This experiment preliminarily proves that reducing the synthesis of NRF2 can reduce the content of rhythm proteins CLOCK and BMAL1 in cells.

2. Effect of different concentrations of NRF2 siRNA on CLOCK, BMAL1 content:

a. the experimental method comprises the following steps:

the experimental groups were as follows (n ═ 4):

(1) DMEM group: culturing NIH/3T3 cells with a normal medium DMEM for 24 h;

(4)50nM NC group: NIH/3T3 cells were transfected with non-fluorescent control NC, i.e., cells were cultured in Opti-MEM medium containing cell transfection reagent liposome 2000(Lipo2000) and 50nM NC for 6h, followed by culturing in DMEM normal medium for 18 h;

(5)100nM NC group: NIH/3T3 cells were transfected with non-fluorescent control NC, i.e., cells were cultured in Opti-MEM medium containing cell transfection reagent liposome 2000(Lipo2000) and 100nM NC for 6h, followed by culturing in DMEM normal medium for 18 h;

(6)200nM NC group: NIH/3T3 cells were transfected with non-fluorescent control NC, i.e., cells were cultured in Opti-MEM medium containing cell transfection reagent liposome 2000(Lipo2000) and 200nM NC for 6h, followed by culturing in DMEM normal medium for 18 h;

(7)50nM SiRNA-1208 group: transfecting NIH/3T3 cells with an RNA interference agent SiRNA-1208, namely culturing the cells in Opti-MEM culture medium containing cell transfection reagent liposome 2000(Lipo2000) and 50nM SiRNA-1208 for 6h, and then culturing the cells in a normal medium DMEM for 18 h;

(8)100nM SiRNA-1208 group: transfecting NIH/3T3 cells with an RNA interference agent SiRNA-1208, namely culturing the cells in Opti-MEM culture medium containing cell transfection reagent liposome 2000(Lipo2000) and 100nM SiRNA-1208 for 6h, and then culturing the cells in a normal medium DMEM for 18 h;

(9)200nM SiRNA-1208 group: NIH/3T3 cells were transfected with the RNA interference agent SiRNA-1208 by culturing the cells in Opti-MEM medium containing Liposome 2000(Lipo2000) as a cell transfection agent and SiRNA-1208 at 200nM for 6h, followed by culturing in DMEM as a normal medium for 18 h.

The sequence of NRF2 siRNA corresponding to each group is shown in the following table 2.

TABLE 2 NRF2 siRNA sequences

b. The experimental results are as follows:

compared with the NC treated group with the corresponding concentration, the expression of NRF2 of the cells after transfection of different concentrations of siRNA-120824 h is obviously reduced, and the expression of CLOCK and BMAL1 is obviously reduced (p is less than 0.05), wherein, the expression of BMAL1 of the cells of the 50nM siRNA-1208 treated group is not obviously different, as shown in figure 5.

c. The experiment summary:

this experiment further demonstrates that reducing the synthesis of NRF2 can reduce the content of the intracellular rhythmic proteins CLOCK, BMAL 1.

Secondly, promoting the decomposition of NRF2 can regulate the content of rhythmic proteins CLOCK and BMAL 1:

1. effect of brucea javanica picrol treatment at different concentrations on cell survival:

a. the experimental method comprises the following steps:

brucellol (Brusatol) has been reported to promote the intracellular degradation of NRF 2. The experiment utilizes Brusatol to promote the decomposition of NRF2 protein of NIH/3T3 cells.

Brusatol is poorly water soluble and needs to be dissolved in dimethyl sulfoxide DMSO. Literature reports that 0.1% DMSO is not damaging to cells, and that this experiment utilizes final concentration of 0.01% DMSO to solubilize Brusatol.

The experimental groups were as follows (n ═ 4):

(1) DMEM group: culturing NIH/3T3 cells with a normal culture medium DMEM for 4 h;

(2) DMSO group: NIH/3T3 cells were cultured for 4h with DMEM containing 0.01% DMSO (Brusatol solvent);

(3)1nM Brusatol panel: NIH/3T3 cells were incubated for 4h with DMEM containing 1nM Brusatol (dissolved in 0.01% DMSO);

(4)10nM Brusatol panel: NIH/3T3 cells were cultured for 4h in DMEM containing 10nM Brusatol (dissolved in 0.01% DMSO);

(5)100nM Brusatol panel: NIH/3T3 cells were cultured for 4h in DMEM containing 100nM Brusatol (dissolved in 0.01% DMSO);

(6)1000nM Brusatol panel: NIH/3T3 cells were cultured for 4h in DMEM containing 1000nM Brusatol (dissolved in 0.01% DMSO).

The cell viability of NIH/3T3 was determined using a cell viability assay kit (Promega, G3580).

b. The experimental results are as follows:

compared with the DMEM group or the DMSO group, the viability of NIH/3T3 cells was not significantly changed after 4h of 1, 10, 100 and 1000nM Brusatol treatment, as shown in FIG. 6.

c. The experiment summary:

these results indicate that treatment with 1-1000nM Brusatol for 4h had no effect on cell survival.

2. Effect of brucea javanica picrol treatment at different concentrations on CLOCK, BMAL1 content:

a. the experimental method comprises the following steps:

the experimental groups were as follows (n ═ 4):

b. The experimental results are as follows:

compared with the DMSO-treated group, the expression of NRF2 in NIH/3T3 cells was significantly reduced after 4h of 1-1000nM Brusatol treatment, indicating that Brusatol promotes the degradation of NRF2 in cells.

Compared with the DMSO-treated group, the expression of CLOCK and BMAL1 was significantly reduced (p <0.05) after 4h treatment with different concentrations of brustol, wherein there was no significant difference in the expression of CLOCK in the cells of the 10nM brustol-treated group, as shown in fig. 7.

c. The experiment summary:

the experiment proves that the content of the rhythm proteins CLOCK and BMAL1 in the cells can be reduced by promoting the decomposition of NRF 2.

Thirdly, increasing the synthesis of NRF2 can regulate the content of the rhythmic proteins CLOCK and BMAL 1:

a. the experimental method comprises the following steps:

gene overexpression techniques are common techniques for increasing intracellular protein synthesis. By transfecting cells with over-expression plasmid pCMV6-Entry-myc-NRF2, which is integrated into the genome of the cells, transcription and translation of the NRF2 protein can be enhanced, thereby increasing synthesis of the NRF2 protein. Wherein myc is a marker for verifying the success or failure of gene overexpression. Myc is a class of small molecule signature genes that encode MYC proteins, which are absent in mammals. After the cells are transfected with an overexpression plasmid pCMV6-Entry-MYC-nrf2, if MYC protein is detected, the gene overexpression is successful.

The experimental groups were as follows (n ═ 4):

(1) DMEM group: culturing NIH/3T3 cells with a normal medium DMEM for 24 h;

(4)500ng NC-myc group: NIH/3T3 cells were transfected with the control plasmid NC-myc over-expressing plasmid, i.e., cells were cultured in Opti-MEM medium containing Liposome 2000(Lipo2000) as a cell transfection reagent and 500ng NC-myc for 6h, and then cultured in DMEM as a normal medium for 18 h;

(5)1000ng NC-myc group: NIH/3T3 cells were transfected with the control plasmid NC-myc over-expressing plasmid, i.e., cells were cultured in Opti-MEM medium containing liposome 2000(Lipo2000) as a cell transfection reagent and 1000ng NC-myc for 6h, followed by 18h DMEM as a normal medium;

(6)2000ng NC-myc group: NIH/3T3 cells were transfected with the control plasmid NC-myc over-expressing plasmid, i.e., cells were cultured in Opti-MEM medium containing liposome 2000(Lipo2000) as a cell transfection reagent and 2000ng NC-myc for 6h, followed by 18h DMEM as a normal medium;

(7)500ng NRF2-myc group: NIH/3T3 cells were transfected with the over-expression plasmid pCMV6-Entry-myc-nrf2, i.e., the cells were cultured in Opti-MEM medium containing liposome 2000(Lipo2000) as a cell transfection reagent and 500ng pCMV6-Entry-myc-nrf2 for 6h, and then cultured in DMEM as a normal medium for 18 h;

(8)1000ng NRF2-myc group: NIH/3T3 cells were transfected with the over-expression plasmid pCMV6-Entry-myc-nrf2, i.e., the cells were cultured in Opti-MEM medium containing liposome 2000(Lipo2000) as a cell transfection reagent and 1000ng of pCMV6-Entry-myc-nrf2 for 6h, and then cultured in DMEM as a normal medium for 18 h;

(9)2000ng NRF2-myc group: NIH/3T3 cells were transfected with the over-expression plasmid pCMV6-Entry-myc-nrf2 by culturing the cells in Opti-MEM medium containing liposome 2000(Lipo2000) as a cell transfection reagent and 2000ng pCMV6-Entry-myc-nrf2 for 6h, followed by 18h in DMEM as a normal medium.

Then, each group of cells was collected, and the expression of biorhythm protein CLOCK, BMAL1 expression, NRF2 protein expression, reference protein β -actin (used in the immunoblotting technique to indicate total protein content) and MYC protein in the cells were detected by the immunoblotting technique.

b. The experimental results are as follows:

the MYC protein in the NRF2-MYC group is detected positively, and the MYC protein in the NC-MYC group and other groups are detected negatively; compared with the NC-myc group with corresponding concentration, the NRF2-myc group NRF2 expression is obviously improved, which indicates that the gene overexpression is successful, and the increase of the synthesis of NRF2 is realized. Compared with the 2000ng NC-myc group, the NIH/3T3 cell CLOCK and BMAL1 expression of the 2000ng NRF2-myc group is remarkably up-regulated (p <0.05), as shown in FIG. 8.

c. The experiment summary:

the experimental result shows that the content of the rhythmic proteins CLOCK and BMAL1 can be increased by increasing the synthesis of NRF 2.

Fourthly, the reduction of the synthesis of NRF2 can regulate the rhythmicity of rhythmic proteins CLOCK and BMAL 1:

a. the experimental method comprises the following steps:

horse serum HS-induced cell synchronization technology is a common technology for researching cell biological rhythm. NIH/3T3 cells generally have no rhythmicity, and horse serum can induce NIH/3T3 cells to synchronize, and the synchronization is represented by fluctuation of the content of rhythmic proteins CLOCK and BMAL1 within 24h, so that the cells present biological rhythms.

The experimental groups were as follows (n ═ 4):

(1) NC + HS group: NIH/3T3 cells were transfected with non-fluorescent control NC, i.e., cells were cultured in Opti-MEM cell transfection medium containing Liposome 2000(Lipo2000) as a cell transfection reagent and NC for 6h, then cultured in DMEM as a normal medium for 18h, and then cultured in DMEM medium containing 50% horse serum for 2 h;

(2) NRF2 siRNA + HS group: NIH/3T3 cells were transfected with NRF2 siRNA by culturing the cells in Opti-MEM medium containing cell transfection reagent liposome 2000(Lipo2000) and NRF2 siRNA for 6h, then culturing in DMEM medium, which is a normal medium, for 18h, and then culturing in DMEM medium containing 50% horse serum for 2 h;

(3) HS group: NIH/3T3 cells were cultured in normal medium DMEM for 24h, and then in DMEM medium containing 50% horse serum for 2 h;

cell samples were taken 1 time every 4h (6 times for CT0, CT4, CT8, CT12, CT16, CT 20) within 24h, and the expression of biorhythm proteins CLOCK, BMAL1 and the expression of endoglin β -actin (used in immunoblotting technique to indicate total protein content) in each group of cells at each time was examined by immunoblotting technique.

b. The experimental results are as follows:

compared to the HS treated group at time point CT8, the NRF2 siRNA + HS treated group had a significant decrease in CLOCK expression at time point CT8 (p < 0.05);

compared to the HS treated group at time point CT4, the NRF2 siRNA + HS treated group CT4 time point BMAL1 expression was significantly increased (p <0.05) as shown in fig. 9.

c. The experiment summary:

this indicates that a reduction in the synthesis of NRF2 can modulate the rhythmicity of the rhythmic proteins CLOCK, BMAL 1.

And fifthly, promoting the decomposition of NRF2 to regulate the rhythmicity of rhythmic proteins CLOCK and BMAL 1:

a. the experimental method comprises the following steps:

the experimental groups were as follows (n ═ 4):

(1) DMSO + HS group: NIH/3T3 cells were cultured with DMEM containing 0.01% DMSO (Brusatol solvent) for 4h, and then cultured with DMEM medium containing 50% horse serum for 2 h;

(2) bruatol + HS group: NIH/3T3 cells were cultured in DMEM containing Brusatol (dissolved in 0.01% DMSO) for 4h, and then in DMEM medium containing 50% horse serum for 2 h;

(3) HS group: NIH/3T3 cells were cultured in normal medium DMEM for 24h, and then in DMEM medium containing 50% horse serum for 2 h.

b. The experimental results are as follows:

both CLOCK and BMAL1 expression were significantly down-regulated (p <0.05) at the brustol + HS treatment group CT4 time point compared to the HS treatment group at the CT4 time point, as shown in fig. 10.

c. The experiment summary:

these results indicate that promoting the decomposition of NRF2 can regulate the rhythmicity of biological rhythm proteins CLOCK and BMAL 1.

As can be seen from the above experiments, the NRF2 protein can regulate the content and rhythmicity of cell biorhythm proteins CLOCK and BMAL1, so that the NRF2 protein can regulate biorhythm.

Example 2:

the adjustment and mechanism research of bortezomib BTZ on biological rhythm aims at: the mouse embryo fibroblast cell line NIH/3T3 cell is taken as a research object, bortezomib BTZ is used for activating NRF2, all-trans retinoic acid ATRA is used for inhibiting the activity of NRF2, and the influence of bortezomib on the content and rhythmicity of biorhythmic protein CLOCK and BMAL1 is researched; the molecular mechanism of regulating biological rhythm by bortezomib is researched by detecting the binding condition of NRF2 protein and Clock and Bmal1 genes.

The procedure and results are shown below.

First, BTZ can regulate the content of rhythmic proteins CLOCK and BMAL 1:

1. effect of different concentrations of BTZ and ATRA treatment on NRF2 activity:

a. experimental methods

After being activated (not necessarily with increased content), the NRF2 protein enters the cell nucleus and is combined with downstream genes to promote the expression of the genes and the synthesis of the protein, thereby increasing the content of the protein downstream of NRF 2.

The literature reports that bortezomib BTZ can enhance the activity of NRF2, and that all-trans retinoic acid ATRA can inhibit the activity of NRF 2. BTZ and ATRA are poorly water soluble and need to be dissolved in dimethyl sulfoxide DMSO. Literature reports that 0.1% DMSO is not damaging to cells, and this experiment uses final concentrations of 0.01% or 0.1% DMSO to dissolve BTZ and 0.1% DMSO to dissolve ATRA.

The experimental groups were as follows (n ═ 4):

experiment A:

(1) 0.01% DMSO group: NIH/3T3 cells were cultured with DMEM containing 0.01% DMSO (BTZ solvent) for 24 h;

(2)5nM BTZ group: cells were cultured for 24h in DMEM containing 5nM BTZ (dissolved in 0.01% DMSO);

(3)50nM BTZ panel: cells were cultured for 24h in DMEM containing 50nM BTZ (dissolved in 0.01% DMSO);

(4)500nM BTZ group: cells were cultured for 24h in DMEM containing 500nM BTZ (dissolved in 0.01% DMSO);

experiment B:

(1) 0.1% DMSO group: NIH/3T3 cells were cultured with DMEM containing 0.1% DMSO (BTZ solvent) for 24 h;

(2)50nM BTZ panel: cells were cultured for 24h in DMEM containing 50nM BTZ (dissolved in 0.1% DMSO);

(3)100 μ M ATRA group: cultured cells containing 100 μ M ATRA (dissolved in 0.1% DMSO) for 24 h;

(4)50nM BTZ + 100. mu.M ATRA group: cells were cultured for 24h in DMEM containing 50nM BTZ and 100. mu.M ATRA (dissolved in 0.1% DMSO);

then, each group of cells was collected, and cytoplasm (Cytoplasmic extract) and nucleus (nuclear extract) were extracted, and the activity of NRF2 was detected using NRF2 activity detection kit (Active Motif, 50296).

b. The experimental results are as follows:

in experiment A, compared with the corresponding 0.01% DMSO treatment group, the activity of NIH/3T3 cytoplasm (p <0.05) and nucleus (p <0.01) NRF2 is remarkably enhanced after 50 and 500nM BTZ treatment for 24 h; treatment with 5nM BTZ did not affect the activity of NRF 2.

In experiment B, after 50nM BTZ treatment for 24h, the activity of NRF2 in nucleus of NIH/3T3 cell is significantly enhanced (p <0.01), and 100 μ M ATRA can inhibit (p <0.01) the enhanced activity of NRF2 caused by BTZ, as shown in FIG. 11.

c. The experiment summary:

this experiment verifies the enhancement of the activity of NRF2 by bortezomib BTZ and the inhibition of the activity of NRF2 by all-trans retinoic acid ATRA.

2. Effect of different concentrations of BTZ on CLOCK, BMAL1 content:

a. the experimental method comprises the following steps:

the experimental groups were as follows (n ═ 4):

then, each group of cells is collected, Cytoplasm (Cytoplasm) and Nucleus (Nucleus) are extracted, and intracellular biorhythmic protein CLOCK, BMAL1 expression, NRF2 protein expression, cytoplasmic reference protein β -actin (used for indicating the content of cytoplasmic protein in immunoblotting technology) expression and nuclear reference protein Histone H3 (used for indicating the content of nuclear protein) expression are detected by immunoblotting technology.

b. The experimental results are as follows:

for cytoplasm, there was no significant change in cellular NRF2 expression after 24h treatment with different concentrations of BTZ compared to the corresponding 0.01% DMSO treatment group; for nuclei, there was no significant change in NRF2 expression in the nuclei of the remaining concentration treated groups, except that the 500nM BTZ treated group significantly reduced NRF2 expression (p < 0.05).

For cytoplasm, there was no significant change in CLOCK, BMAL1 expression after 24h treatment with different concentrations of BTZ compared to the corresponding 0.01% DMSO treatment group; for nuclei, there was a significant upregulation of CLOCK expression (p <0.05) after 50, 500nM BTZ treatment with no significant change in BMAL1 expression, as shown in figure 12.

c. The experiment summary:

these results indicate that BTZ can modulate the content of the rhythmic proteins CLOCK, BMAL1, which can be independent of the NRF2 content.

3. Effect of BTZ and ATRA on CLOCK, BMAL1 content:

a. experimental methods

The experimental groups were as follows (n ═ 4):

(3)100 μ M ATRA group: cells were cultured for 24h with DMEM containing 100 μ M ATRA (dissolved in 0.1% DMSO);

then collecting each group of cells, extracting cytoplasmic protein (Cytoplasm) and nucleoprotein (nucleous), detecting expression of biorhythmic protein CLOCK, BMAL1, NRF2 protein, cytoplasmic reference protein β -actin (used for indicating the content of cytoplasmic protein in immunoblotting technology) and nuclear reference protein Histone H3 (used for indicating the content of nuclear protein) in the cells by immunoblotting technology.

b. The experimental results are as follows:

for cytoplasm or nuclei, there was no significant change in cellular NRF2 expression after 24h treatment with different concentrations of BTZ or ATRA compared to the corresponding DMSO-treated groups.

For cytoplasm, 50nM BTZ treatment significantly upregulated CLOCK, BMAL1 expression compared to DMSO treated group (p < 0.01); for nuclei, 100 μ M ATRA treatment alone did not affect intra-nuclear CLOCK, BMAL1 expression, but inhibited BTZ-induced up-regulation of expression (p <0.05), as shown in fig. 13.

c. The experiment summary:

these results further indicate that BTZ can regulate the content of biorhythm proteins CLOCK, BMAL1 in a manner independent of the change in NRF2 content.

Secondly, BTZ can regulate the rhythmicity of rhythmic proteins CLOCK and BMAL 1:

a. experimental methods

The experimental groups were as follows (n ═ 4):

(1) DMSO group: NIH/3T3 cells were cultured with DMEM containing 0.1% DMSO (a solvent for BTZ and ATRA) for 24h, and then cultured with DMEM medium containing 50% horse serum for 2 h;

(2) BTZ group: NIH/3T3 cells were cultured with DMEM containing 50nM BTZ (dissolved in 0.1% DMSO) for 24h, then with DMEM medium containing 50% horse serum for 2 h;

(3) BTZ + ATRA group: NIH/3T3 cells were cultured with DMEM containing 50nM BTZ and 100. mu.M ATRA (dissolved in 0.1% DMSO) for 24h, followed by 50% horse serum in DMEM for 2 h.

Cell samples were taken 1 time every 4h (6 times for CT0, CT4, CT8, CT12, CT16, CT 20) over 24h, and the expression of the cell biorhythmic proteins CLOCK, BMAL1 and the expression of the internal reference protein GAPDH (used in immunoblotting techniques to indicate total protein content) were examined for each group at each time by immunoblotting technique.

b. The experimental results are as follows:

as shown in fig. 14, the level of CLOCK expression was significantly increased in the BTZ-treated group compared to the DMSO-treated group at time points CT4, CT12 (p <0.05) and was inhibited by ATRA treatment.

Compared to the DMSO-treated group, the BTZ-treated group had significantly increased expression levels of BMAL1 at the CT4 time point (p <0.05) and could be inhibited by ATRA treatment.

In addition, the ATRA-treated group had significantly reduced expression levels of BMAL1 at CT0 (p <0.05) compared to the DMSO-treated group.

c. The experiment summary:

these results indicate that BTZ can regulate the rhythmicity of the biorhythmic proteins CLOCK, BMAL 1.

Third, BTZ regulates Clock, Bmal1 by promoting NRF2 binding to Clock, Bmal1 genes:

a. the experimental method comprises the following steps:

the experimental groups were as follows (n ═ 4):

experiment A:

experiment B:

then, each group of cells was collected, nucleoproteins were extracted, and binding of NRF2 to Clock and Bmal1 genes was detected by chromatin co-immunoprecipitation.

b. The experimental results are as follows:

as shown in figure 15, 50, 500nM BTZ treatment for 24h enhanced nuclear NRF2 binding to the Clock, Bmal1 gene (p <0.05) compared to the 0.01% DMSO treated group.

Compared with the 50nM BTZ group, the 50nM BTZ + 100. mu.M ATRA group NRF2 showed significantly reduced binding to the Clock gene (p <0.01), and NRF2 showed reduced binding to Bmal1 gene with no statistical difference.

c. The experiment summary:

these results indicate that BTZ regulates Clock, Bmal1 by promoting the binding of NRF2 to the Clock, Bmal1 genes.

From the above experimental results, it can be seen that bortezomib BTZ promotes NRF2 to combine with Clock and Bmal1 genes by activating NRF2 protein, and regulates the content and rhythmicity of rhythmic proteins Clock and Bmal1, thereby exerting its function of regulating biological rhythm.

The core content of the technical scheme required by the invention is as follows: the bortezomib BTZ activates NRF2 protein, promotes NRF2 to be combined with genes related to biological rhythms, and regulates the content and rhythmicity of the proteins related to the biological rhythms, so that the bortezomib BTZ plays a role in regulating the biological rhythms. The most important technical means are as follows: (1) modulation of biological rhythms by activation of NRF2 protein; (2) the biological rhythm is regulated by promoting the combination of NRF2 protein and biological rhythm related gene.

Meanwhile, three possible similar ways are possible to achieve the corresponding purposes, specifically as follows:

1.NRF2 is activated by other means.

The ways to activate NRF2 include NRF2 agonists, release of KEAP-1 inhibition of NRF2 protein, promotion of NRF2 protein phosphorylation, increase of NRF2 content, etc.

Thus, such alternative technologies include, but are not limited to, the following 4:

(1) NRF2 agonists other than the present invention (bortezomib) were administered to activate NRF2 protein. Such as tert-butyl hydroquinone tBHQ, dithiacyclopentadiene thioketone compound D3T, triterpenoid compound CDDO-Im, etc.;

(2) the KEAP-1 protein inhibitor was administered to release the inhibition of NRF2 protein by KEAP-1 (the KEAP-1 protein typically binds to NRF2 to prevent NRF2 from being activated) to indirectly activate NRF2 protein. Such as ML334(LH601A), Keap1-Nrf2 IN 1(compound35), etc.;

(3) administration of a phosphokinase activator promotes phosphorylation of NRF2 protein, thereby activating NRF2 protein. Such as phosphatidylinositol-3-kinase PI3K agonists VEGF, FGF, PDGF, IGF-1, and Protein Kinase C (PKC) agonists Myristica fragrans acetate PMA, and the like;

(4) in addition to the invention (by cell transfection of the overexpression plasmid pCMV6-Entry-myc-NRF2), the invention also provides a way to increase the NRF2 content, such as increasing the level of NRF2 mRNA, reducing the degradation of NRF2 protein, and the like.

2. Binding of NRF2 protein to Clock, Bmal1 gene was promoted by other means.

Such alternative techniques include, but are not limited to, modifications to the environmental conditions required for protein to gene binding, such as optimizing solution pH, optimizing the relevant enzyme activity, optimizing binding cofactor concentration, and the like.

3. Through NRF2 protein to combine with other genes related to biological rhythm, so as to regulate biological rhythm.

The biological rhythm related genes other than those of the present invention (Clock, Bmal1 gene) include, but are not limited to, Cry1, Cry2, Per1, Per2, Per3, Rev-reb α, Rev-reb β, Ror α, etc.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Chinese people liberation army 63919 army

Application of <120> NRF2 protein in preparing medicine for regulating biological rhythm

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<170>SIPOSequenceListing 1.0

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Ser Arg Glu Val Phe Asp Phe Ser Gln Arg Arg Lys Glu Tyr Glu Leu

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Glu Lys Gln Lys Lys Leu Glu Lys Glu Arg Gln Glu Gln Leu Gln Lys

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Glu Gln Glu Lys Ala Phe Phe Thr Gln Leu Gln Leu Asp Glu Glu Thr

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Gly Glu Phe Leu Pro Ile Gln Pro Ala Gln His Thr Gln Ser Glu Thr

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Ser Gly Ser Ala Asn Tyr Ser Gln Val Ala His Ile Pro Lys Ser Asp

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Ala Leu Tyr Phe Asp Asp Cys Met Gln Leu Leu Ala Gln Thr Phe Pro

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Phe Val Asp Asp Asn Glu Val Ser Ser Ala Thr Phe Gln Ser Leu Val

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Pro Asp Ile Pro Gly His Ile Glu Ser Pro Val Phe Ile Ala Thr Asn

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Leu Asp Gly Met Gln Gln Asp Ile Glu Gln Val Trp Glu Glu Leu Leu

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Ser Ile Pro Glu Leu Gln Cys Leu Asn Ile Glu Asn Asp Lys Leu Val

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Glu Thr Thr Met Val Pro Ser Pro Glu Ala Lys Leu Thr Glu Val Asp

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Asn Tyr His Phe Tyr Ser Ser Ile Pro Ser Met Glu Lys Glu Val Gly

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Ile Leu Ser Thr Glu Asp Pro Asn Gln Leu Thr Val Asn Ser Leu Asn

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Ser Asp Ala Thr Val Asn Thr Asp Phe Gly Asp Glu Phe Tyr Ser Ala

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Phe Ile Ala Glu Pro Ser Ile Ser Asn Ser Met Pro Ser Pro Ala Thr

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Ser Val Ala Ser Pro Glu His Ser Val Glu Ser Ser Ser Tyr Gly Asp

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Gly Asp Met Val Gln Pro Leu Ser Pro Ser Gln Gly Gln Ser Thr His

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Val His Asp Ala Gln Cys Glu Asn Thr Pro Glu Lys Glu Leu Pro Val

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Tyr Leu Glu Val Phe Ser Met Leu Arg Asp Glu Asp Gly Lys Pro Tyr

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Claims

Use of NRF2 protein for the manufacture of a medicament for modulating biological rhythms.
2. The use of claim 1, wherein the amino acid sequence of NRF2 protein is shown as sequence 1 in the sequence table.
3. Use according to claim 2, characterized in that the modulation of the biorhythm is achieved in two ways:

1) modulation of biological rhythms is achieved by activation of NRF2 protein;

2) the regulation of biological rhythm is realized by promoting the combination of NRF2 protein and genes related to biological rhythm.
4. The use of claim 3, wherein the activation of NRF2 protein and the promotion of the binding of NRF2 protein to genes associated with biological rhythms are both achieved by NRF2 agonists.
5. The use of claim 4, wherein the NRF2 agonist is bortezomib.