CN115737612A - Use of SLC3A2 inhibitor in preparing anti-inflammatory drug - Google Patents
Use of SLC3A2 inhibitor in preparing anti-inflammatory drug Download PDFInfo
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Abstract
The invention provides an application of an SLC3A2 inhibitor in preparing an anti-inflammatory medicament and an application of honokiol as an SLC3A2 inhibitor. The invention proves that honokiol can be used as an SLC3A2 inhibitor for the first time, and the proteasome dependent degradation of honokiol is enhanced through direct interaction with SLC3A 2; the SLC3A2 inhibitor can influence the transportation of leucine in THP-1 cells and inhibit the activation of mTORC1, so that the activation of NLRP3 inflammasome is inhibited, the anti-inflammatory effect is realized, the obvious treatment effect is realized on acute inflammatory diseases such as septicemia and ulcerative colitis, and a new way is provided for the screening and research of anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to an application of an SLC3A2 inhibitor in preparation of an anti-inflammatory drug.
Background
NLRP3 (Nod-like receptor pyridine-binding 3) inflammasomes are an important component of the innate immune system, often involved in immune responses to bacteria, viruses, fungi, and parasites. It can be activated by a variety of stimuli, including exogenous (pathogen-associated molecular patterns including microbial nucleic acids, bacterial secretion systems, and microbial cell wall components) and endogenous (damage-associated molecular patterns such as ATP and uric acid crystals). Once activated, NLRP3 can induce the production of an adapter ASC and the production of active caspase-1 by effector procaspase-1 of NLRP3 inflammasome, which then converts the cytokine precursors pro-IL-1 β and pro-IL-18 to mature, biologically active IL-1 β and IL-18, respectively, triggering a series of inflammatory responses that ultimately lead to focal cell death.
Magnolia officinalis is a traditional medicine, is mainly used for treating gastrointestinal diseases in Asian countries, and is originally mentioned in the classical Chinese herbal medicine Shennong Ben Cao Jing (Shennong's herbal medicine) compiled in the 1 st or 2 nd century of the public Yuan. Researches show that the magnolia officinalis and a preparation containing the magnolia officinalis (such as Pingwei san, which is seen in the first 1107 years of the Bu Yuan) can treat ulcerative colitis by repairing colonic mucosal injury, regulating immune phagocytosis, improving inflammatory response and the like. Honokiol (HK) is a natural biphenyl neolignan, is the main active component of Magnolia officinalis, and can be widely used in Chinese herbal medicine and health food material. HK has been reported to have anti-inflammatory activity by inhibiting the release of pro-inflammatory cytokines through modulation of various inflammatory factors, such as the transcription factors Klf4 or NF-. Kappa.B. It has also been shown to modulate the PI3K/Akt pathway and phosphorylation of p38, ERK1/2 and JNKs kinases. Recently, researchers have demonstrated that HK can inhibit activation of NLRP3 inflammasome by Nrf2 activation, down-regulation of TXNIP, and enhancement of sirtuin 1/autophagy axis activation. Although studies have reported that HK exhibits anti-inflammatory activity by inhibiting NLRP3 inflammasome activation, the route by which HK inhibits NLRP3 inflammasome activation is not yet clear. Because of the close relationship between NLRP3 inflammasome and metabolism and neurodegenerative diseases, the regulatory factor for directly or indirectly regulating the activation of NLRP3 inflammasome draws extensive attention, so that the discovery of the novel NLRP3 inflammasome regulatory factor by taking HK as a probe has important significance.
SLC3A2 is called the 4F2 heavy chain and is a type II membrane protein. SLC3A2 dimerizes several nutrient transporter light chains (e.g., SLC7A5, also known as LAT 1) as a partner, localizing it to the plasma membrane. The SLC3A2/SLC7A5 heterodimer is reported to be responsible for the exchange of intracellular glutamine and extracellular L-leucine, which is critical for the activation of mTORC 1. mTORC 1-induced hexokinase 1-dependent glycolysis is reported to provide an important metabolic mechanism for NLRP3 inflammasome activation. Thus, inhibition of mTORC1 can inhibit activation of NLRP3 inflammasome. However, it is not clear whether SLC3A2 is involved in activation of NLRP3 inflammasome, and to date, there has been no report of a direct interaction of small molecules with SLC3A2. And at present, no report proves the targeting of the HK to the SLC3A2, and no report proves the correlation between the SLC3A2 and the NLRP 3.
Disclosure of Invention
The invention aims to provide application of honokiol as an SLC3A2 inhibitor and application of the SLC3A2 inhibitor in preparing anti-inflammatory drugs.
The invention provides an application of an SLC3A2 inhibitor in preparing an anti-inflammatory medicament.
Further, the SLC3A2 inhibitor is an agent for reducing the SLC3A2 content in the body.
Further, the SLC3A2 inhibitor is a reagent for inhibiting the expression of the SLC3A2 gene or a reagent for promoting the degradation of the SLC3A2 protein, and is preferably a reagent for promoting the degradation of the SLC3A2 protein.
Further, the above SLC3A2 inhibitor is an agent which attenuates the ability of a cell to take up leucine.
Further, the above SLC3A2 inhibitor is an agent for inhibiting the activity of mTOR.
Further, the above SLC3A2 inhibitor is an agent for inhibiting NLRP3 activity.
Further, the anti-inflammatory agent is a drug for treating acute inflammatory diseases.
Further, the above-mentioned drug is a drug for treating sepsis and/or ulcerative colitis.
The invention also provides the use of honokiol as an SLC3A2 inhibitor.
Further, the SLC3A2 inhibitor is an agent for reducing the content of SLC3A2 in the body or an agent for inhibiting the activity of SLC3A2, and preferably an agent for directly acting on SLC3A2 protein to reduce the content of SLC3A2 in the body.
The invention has the beneficial effects that: the invention proves that honokiol can be used as an SLC3A2 inhibitor for the first time, and the proteasome dependent degradation of honokiol is enhanced through direct interaction with SLC3A 2; the SLC3A2 inhibitor can influence the transportation of leucine in THP-1 cells and inhibit the activation of mTORC1, so that the activation of NLRP3 inflammasome is inhibited, the anti-inflammatory effect is realized, the obvious treatment effect is realized on acute inflammatory diseases such as septicemia and ulcerative colitis, and a new way is provided for the screening and research of anti-inflammatory drugs.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
Figure 1 is the process of honokiol inhibition of NLRP3 inflammasome initiation and activation. (A-B) LPS-induced THP-1 cells were treated with HK for 40min and then stimulated with 5mM ATP for 1h. The supernatants were analyzed for IL-1. Beta. (A) and TNF-. Alpha. (B) release by ELISA; n =3, mean ± SD. (C) The medium Supernatants (SN) and cell extracts (Input) were analyzed by immunoblotting for the expression of IL-1. Beta. And caspase-1. (D) Measuring the release of LDH in the supernatant with or without ATP stimulation after 40min of LPS-induced THP-1 cells treated with different concentrations of HK (5, 10 and 20 μ M); n =3, mean ± SD. (E-H) LPS-induced BMDMs were treated with different concentrations of HK (5 and 10. Mu.M), stimulated with nikkomycin (E), ATP (F), MSU (G), alum (H) and IL-1. Beta. In the supernatant, and analyzed by ELISA; n =3, mean ± SD. (I) Cell lysates were analyzed by immunoblotting for caspase-1 expression. (J-L) THP-1 cells were treated with HK for 40min, then primed with LPS for 3h and stimulated with ATP. Detecting IL-1 beta (J) and TNF-alpha (K) in the supernatant by an ELISA method; n =3, mean ± SD. SN and Input (I) were analyzed by immunoblotting and statistics were tested by one-way anova with P <0.05, P <0.01, P <0.001.
Figure 2 is that honokiol inhibits ASC oligomerization. (A) Representative immunofluorescence images of ASC oligomerization of LPS-mediated THP-1 cells in the presence or absence of HK for 40min, ATP stimulated for 1h. Scale bar, 100 μm; n =3; (B) counting the percentage of ASC spot cells to the total number of cells; n =3, mean ± SD; (C) Immunoblot analysis of oligomerization of ASC in lysates of HK (5, 10, and 20 μ M) -treated THP-1 cells. Data analysis was checked using one-way analysis of variance.
Figure 3 is a target SLC3A2 of potential effect of honokiol found by a target stability technology and a proteomics technology of drug affinity targeting reaction. (A) PMA differentiation and LPS-induced THP-1 cell lysates were incubated with different concentrations (10, 100 and 1000 μ M) of HK for 45min, followed by hydrolysis with pronase for 30min and finally with cocktail to stop the hydrolysis. Protein samples were analyzed by SDS-PAGE and stained with Coomassie blue. Red arrows indicate protective proteins around-100 kDa; (B) a honokiol-treated protein in THP-1 cells. Volcanoes showed that a total of 109 proteins were down-regulated and 21 proteins were up-regulated by HK treatment compared to ATP-induced group in LPS-initiated THP-1 cells. Downregulated protein (blue) >1.5 fold, P value <0.001, upregulated protein (red) >1.5 fold, P value <0.001, invariant protein expressed in grey; (C) Thermographic analysis of differentially expressed proteins was validated in proteomics analysis (43 representative proteins were presented). Red for up-regulated protein and green for down-regulated protein. (D) The venn diagram shows the intersection of the 1 (i), 2 (iii) band proteins with specific regulatory proteins after HK treatment in proteomic analysis (ii). The graph was generated with a >1.5 fold change in significant protein and a p value of 0.001 or less. (E, H) DARTS assay for target validation. THP-1 cell lysate (E) or purified His-SLC3A2 protein (H) of LPS primers were incubated with different concentrations of HK and prose; (F, G) HK target binding was analyzed by CETSA method. HK blocks the precipitation of SLC3A2 with temperature dependence (F) and concentration dependence (G). (I, J) in vitro TSA. HK blocks the precipitation of purified His-SLC3A2 protein, whose mechanism of action is temperature-dependent (I) and concentration-dependent (J).
Figure 4 is a graph showing that silencing expression of SLC3A2 inhibits activation of NLRP 3. (A) THP-1 cells stably transfected with a control sequence (sh-NT) and sh-SLC3A 2. Detecting the expression of SLC3A2 in the cell by western blot; (B-D) sh-NT and sh-SLC3A2 THP-1 cells were differentiated with PMA, primed with LPS, treated with honokiol for 40min, and then stimulated with ATP. Detecting IL-1 beta in the supernatant by an ELISA method; n =3, mean ± SD. Statistical analysis using two-way ANOVA analysis,. P <0.01 (B). Cell extracts (input) were analyzed for SLC3A2, and Supernatants (SN) were analyzed by immunoblotting for IL-1 β and caspase-1 levels (C). Cells were lysed with NP-40 lysate and soluble protein was collected and cross-linked with DSS. Western blots detected the level of polymerization (D) of ASC, SLC3A2, IL-1. Beta. And caspase-1.
Figure 5 is a graph of the results of honokiol degradation by promoting the ubiquitin proteasome of SLC3 A2; (A-B) LPS-induced THP-1 cells were treated with honokiol and then stimulated with ATP. The expression of SLC3A2 is detected by adopting a western blot (A) method, and the expression of SLC3A2 is detected by adopting a qPCR method. LPS-treated THP-1 cell mRNA levels were normalized to 1; n =3, mean ± SEM; (C) Proteasome inhibitor MG132 (10. Mu.M) or the autophagy inhibitor 3-MA (5 mM) 1h were treated before LPS treatment, LPS-mediated THP-1 cells were not stimulated by LPS (LPS only) or ATP (no or absent HK), and cell lysates were analyzed for SLC3A2 content by immunoblotting. (D) MG132 was pretreated for 1h prior to LPS treatment, then incubated with HK, then stimulated with ATP, and then analyzed for SLC3A2 localization using immunofluorescence. Scale bar is 10 μm; (E) Cell lysates were analyzed by immunoblotting for SLC3A2 following induction with HK or HK and PYR41 (10. Mu.M).
Fig. 6 is a graph of the results of honokiol inhibiting activation of NLRP3 inflammasome by blocking leucine uptake and mTORC1 activation; (A) UFLC-MS/MS analysis N 15 -leucine uptake into THP-1 cells; n =3, mean ± SD; (B) Analysis of N 15 -leucine entry into lysosomes; n =3, mean ± SD; (C) LPS-induced THP-1 cells were treated with HK (5, 10 and 20 μ M), respectively, and then stimulated with ATP. Immunofluorescence Using SLC3A2, LAMP2 and DAPICells were analyzed by light. Scale bar is 10 μm; (D-F) LPS-induced THP-1 cells were treated with HK or MHY1485 (10 μ M), respectively, for 40min, followed by stimulation with ATP. Cells were lysed, immunoblotted with the indicator antibody (D), levels of phosphorylated-P70S 6K1 (P-P70S 6K 1) relative to P70S6K1 (E) were quantified, and IL-1. Beta. In the supernatant was detected by ELISA; n =4, mean ± SD (F). (G) ASC polymerization was detected in LPS-mediated THP-1 cells after treatment with HK or rapamycin (10 nM). The statistical data is tested by adopting single-factor analysis of variance; * p is a radical of<0.05，**p<0.01，***p<0.001。
Figure 7 is a graph of the results of HK treatment that increased survival of mice sepsis and inhibited DSS-induced ulcerative colitis. (a-C) HK effectively inhibited the lps-induced septic shock mouse model, n =6, mean ± SD. Serum IL-1 β (A), TNF- α (B) levels and mouse survival curves (C); (D-I) HK was effective in inhibiting dss-induced ulcerative colitis, n =6, mean. + -. SD. Percent body weight change during dosing (D); DAI was derived from the average sum of weight loss score, stool consistency, major bleeding and coat color status (E). Colon length is measured between the ileum-cecum junction and the proximal rectum (F). Colon sections were stained with H & E. Expression of SLC3A2 in colon tissue extracts was analyzed by immunoblotting (H) and expression of IL-1 β by ELISA (I), using one-way anova test, P <0.05, P <0.01, P <0.001.
Detailed Description
The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.
The experimental method and operation involved in the present invention are as follows:
(1) THP-1 and BMDMs cell culture and stimulation
Human THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The differentiated THP-1 cells were obtained by co-culturing with 100ng/mL PMA for 24h. BMDMs were derived from C57BL/6 mouse femurs and tibias, cultured in DMEM medium supplemented with 10% fetal bovine serum, and supplemented with 30% L929 culture supernatant.
Induction of activation of NLRP3 inflammasome, 2X 10 5 The differentiated THP-1 cells or BMDMs were cultured in 48-well plates for 24 h/mL. After that time, the user can use the device,the supernatant was discarded, and the cells were stimulated with opti-MEM medium containing 1. Mu.g/mL LPS for 3 hours, followed by addition of a certain concentration of honokiol or solvent for 40 minutes. Cells were treated with 150. Mu.g/mL MSU, 300. Mu.g/mL alum for 6h, 10. Mu.M nystatin for 40min or 5mM ATP and 1h. AIM2 and NLRC4 inflammasomes were transfected with 500ng/mLpoly (dA: dT) and 250ng/mL flagellin, respectively, using Lipofectamine 2000 reagent according to the manufacturer's instructions.
(2) Cytokine secretion assay
ELISA kit detects the concentration of TNF-alpha and IL-1 beta in cell-free culture supernatant. The detection step was performed according to the manufacturer's instructions.
(3) Immunofluorescence
After cell induction was complete, the supernatant was discarded, the cells were washed with pre-cooled PBS, fixed in ice-cold methanol at-20 ℃ for 20min, washed with PBST for 5min, and blocked with 0.5% bovine serum albumin (w/v, diluted in PBST) for 1h. Followed by overnight incubation with antibody. Then washed with PBST, treated for 1h with secondary antibody diluted 1.5% bovine serum albumin while staining nuclei with 4',6' -diamino-2-phenylindole (DAPI). Cells were then observed and imaged with a fluorescence microscope (zeiss Axiovert 200, shanghai, china).
(4) ASC oligomerization
The cells were lysed with 300. Mu.L NP-40 lysis buffer, the lysate incubated on a shaker at 4 ℃ for 30min, centrifuged at 6000 Xg for 15min,4 ℃. After removal of the supernatant, insoluble cell debris was incubated in 2mm DSS (Sigma-Aldrich, USA) at 37 ℃ for 30 minutes, centrifuged at 6000 Xg for 15min at 4 ℃, and the pellet resuspended in 30. Mu.L of 1 Xloading buffer for western blot detection.
(5) Drug Affinity Reaction Target Stability (DARTS)
PMA-induced differentiation and LPS-primed THP-1 cells were washed 2 times with pre-chilled PBS and 500. Mu.L of pre-chilled NP40 lysate buffer was added. The cells were scraped off with a cell scraper, ice-cooled for 30 minutes, and centrifuged (13000 Xg, 4 ℃,15 minutes). The supernatant was then divided into 99 μ L aliquots and loaded into EP tubes. Add 1. Mu.L DMSO solution or honokiol and incubate for 45 minutes at room temperature. In addition to the control group, 10. Mu.L pronase (1. Mu.g pronase per 500. Mu.g cell lysate) was added to each of the other groups, incubated at room temperature for 30 minutes, stopped by adding 10. Mu.L of 20 XPase inhibitor cocktail, treated at room temperature for 15 minutes, and treated at 100 ℃ with 30. Mu.L of 5 XPading buffer for 10 minutes. And detecting the sample by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), dyeing for 30 minutes by room temperature Coomassie brilliant blue, eluting by stripping liquid, observing a specific protection band, and detecting the type of the protein in the band by mass spectrometry.
(6) Thermal displacement experiment
To test the thermal stability of the target protein, THP-1 cells of LPS1 were washed twice with pre-cooled PBS. Then, the cells were gently scraped with a cell scraper, and freeze-thawed repeatedly three times with liquid nitrogen. After centrifugation at 2000 Xg for 20min at 4 ℃ the supernatant was added in equal amounts to a PCR tube at a total volume of 49. Mu.L, HK or DMSO was added at 1. Mu.L, and gently mixed. The reaction mixture was heated in a BIO-RAD T100TM thermocycler at the indicated temperature for 3 minutes, then at room temperature for 3 minutes, and treated with 5 × loading buffer 12.5 μ L at 100 ℃ for 10 minutes. WB detects changes in the protein of interest.
(7)N 15 Leucine uptake
LPS-primed THP-1 cells were plated in 6-well plates (2X 10) 5 cells/mL) was allowed to act in the presence of honokiol for 40 minutes, ATP (5 mM) stimulated with 1h. The supernatant was discarded, washed twice with HBSS buffer (Melphalan, china) and starved for 50 minutes by adding HBSS buffer again, then starved cells were treated with 10mM glutamine 1h, HBSS washed twice, and then treated with a solution containing 25. Mu.M N 15- HBSS treatment of L-leucine for 10 min. Cells were washed with pre-chilled PBS, resuspended at 200. Mu.L PBS, and sonicated for 5min.13400 Xg ultracentrifugation for 15min, collecting supernatant, UFLC-MS/MS detecting N 15- L-leucine concentration. Detection of N in lysosomes 15- When the content of L-leucine is high, lysosome is extracted by a lysosome extraction kit, and then N is detected by ultrasonic lysosome cracking 15- L-leucine content.
(8) Animal experiments
Male C57BL/6 mice (20-22 g) at 8 weeks of age, supplied by Beijing Huafukang Biotechnology, inc. All mice were housed in a university of Sichuan laboratory animal center-specific pathogen-free animal facility, and animal experiments were conducted according to animal experimental protocols approved by the ethical committee for animal experiments in the national institute of emphasis laboratory for biotherapy in Sichuan university.
To induce cytokine secretion and animal survival in vivo, adult mice were intraperitoneally injected with LPS (20 mg/kg) or LPS + HK (35 mg/kg and 70mg/kg, oral), respectively. And collecting a serum sample after 6h, and detecting the cytokine by an ELISA method. To study survival time, the status of mice was observed every 12h after induction and the number of deaths recorded.
Mice were fed 2.5% (w/v) DSS (MW 3.6-50, 000Da, MP biomedicals, inc Irvine, CA, USA) for 10 days, and were free to drink, inducing experimental colitis. These mice were carefully monitored daily to confirm that they took approximately equal amounts of DSS-containing water. To investigate the preventive effect of HK on colitis, mice were randomly divided into 4 experimental groups (n = 6). In the untreated group, mice normally drink water and the control solvent is orally taken 1 time a day; model group mice were orally administered control solvent daily; in the HK (35 and 70 mg/kg) groups, mice were fed DSS water and given honokiol (35 mg/kg) or (70 mg/kg/day) orally. From the modeling date, the conditions of the feeding, the activity and the hair of the mouse are observed every day, the weight of the animal is weighed, the fecal characters and fecal occult blood and bleeding conditions of the mouse are observed, and the severity of the colitis is evaluated. Disease activity condition is scored, disease Activity Index (DAI) score = weight loss score + stool trait score + stool occult blood score + hair color status score, and disease activity index of each mouse is calculated and is totally 16 points. Briefly, the calculation was done using the following parameters a) diarrhea (0 point = normal, 2 points = loose stool, 4 points = watery diarrhea); b) Stool blood (0 point = no bleeding, 2 points = minor bleeding, 4 points = major bleeding); c) Weight loss scores (0 point = no weight loss, 1 point = weight loss 1% -5%,2 point = weight loss 6% -10%,3 point = weight loss 11% -15%,4 point = weight loss greater than 15%); d) The hair color status (0 point = normal, 1 point = hair shrug, 3 points = hair rising and dizzy, 4 points = standing still and dizzy). After the experiment was completed, the mice were sacrificed and the colon was isolated. Colon length is measured from the ileocecal junction to the proximal end of the rectum. Colon tissue (about 10 mg) was ground and washed with 200 μ L of pre-cooled PBS and tested for IL-1 β levels by ELISA, or ground and added to RIPA lysed sample supernatant and tested for SLC3A2 expression by western blot.
Experimental example 1 HK inhibits the initiation and activation process of NLRP3 inflammasome
To test whether HK affects the activation of NLRP3 inflammasome, we first examined the effect of HK on IL-1 β secretion, which is considered to be a marker of NLRP3 inflammasome activation in THP-1 cells. We pre-treated LPS-primed THP-1 cells with HK for 40min, followed by treatment with the NLRP3 inflammasome activator ATP. The results showed that HK concentration-dependently inhibited the maturation of IL-1 β, but had no effect on TNF- α production or on the expression of IL-1 β precursor (p 31) (FIGS. 1A-1C). At the same time, HK concentration-dependently blocked ATP-induced cleavage of caspase-1 to p20, while intracellular caspase-1 precursor levels were unchanged (p 45) (FIG. 1C). These results indicate that HK can inhibit the maturation of IL-1 β by inhibiting LPS-mediated activation of the NLRP3 inflammasome in THP-1 cells.
After NLRP3 inflammasome activation, cells are regulated by gasdermin D, forming pores in the plasma membrane, leading to inflammasome-related cell death-pyro death. The release of Lactate Dehydrogenase (LDH) was then detected and the effect of HK on NLRP3 inflammasome-dependent apoptosis was studied. LDH release experiments showed that ATP treatment could significantly induce THP-1 cell death (fig. 1D). Consistent with the inhibitory effect of HK on NLRP3 inflammasome activation, HK can significantly reduce ATP-induced apoptosis. To test whether HK is a broad-spectrum inhibitor of NLRP3 inflammasome, we used other NLRP3 agonists in BMDMs. The results showed that HK blocked the activation of NLRP3 inflammasome induced by Nihonisin, ATP, alum and monosodium urate crystals (MSU) (FIGS. 1E-1I). Indicating that HK is a broad inhibitor of NLRP 3.
Since inhibition of NF-. Kappa.B activation and TNF-. Alpha.production have been found in the anti-inflammatory activity of HK, we sought to determine whether HK had an effect on the initiation of LPS-induced activation of NLRP3 inflammasome. Consistent with previous studies, our data also show that HK can block the start-up process when it is added before the start signal (fig. 1J-1L).
The above results demonstrate that HK can inhibit the secretion of IL-1 beta and the activation of caspase-1 by inhibiting the initiation and activation process of NLRP3 inflammasome, and achieve anti-inflammatory effects.
Experimental example 2 HK inhibits oligomerization of ASC
Since ASC oligomerization promotes caspase-1 activation, which is considered to be a marker for NLRP3 inflammasome activation, we further investigated the effect of HK on ASC oligomerization. The results show that HK can inhibit ASC oligomerization during NLRP3 inflammasome activation (fig. 2A-2C), suggesting that HK acts on the upstream signaling pathway of ASC oligomerization, further confirming the inhibitory effect of HK on NLRP3 inflammasome activation.
Example 3 HK inhibition of NLRP3 inflammasome by direct targeting of SLC3A2
To determine potential targets for the inhibitory activity of HK on NLRP3 inflammasome, a Drug Affinity Reaction Target Stability (DARTS) assay was used. The principle of this technique is that the protein structure is stable after drug conjugation, resulting in a target protein that is resistant to protease degradation. DARTS detection revealed two strong protective bands around the 100-kDa MW marker in the proteolytic extract of HK-treated cells (FIG. 3A, red arrows). Analysis of the protective bands by LC/MS identified a total of 179 proteins in band 1 and 330 proteins in band 2.
With such enormous data, the target for HK determination remains very solvent-free. Then further using proteomic analysis, proteins specifically altered after HK treatment in THP-1 cells were determined. A total of 130 proteins were specifically regulated by HK (> 1.5FC, P.ltoreq.0.05) (FIG. 3B,3C and FIG. S2 and data set S3). Analysis of proteins detected in Band 1 and proteins specifically regulated in proteomic analysis found that two proteins overlap: SLC3A2 and ALCAM (fig. 3D). The same strategy is adopted for Band 2 and specific regulatory protein in proteomics analysis, and 3 proteins are found to be overlapped, namely SLC3A2, ANKHD1 and SLC1A5. Finally, interestingly, SLC3A2 was found to be the only one of Band 1, band 2 and specific regulatory protein after HK treatment in proteomic analysis (fig. 3D). The reason SLC3A2 was detected in both Band 1 and Band 2 may be due to glycosylation of SLC3A2.
To verify that SLC3A2 is the molecular target for HK, we performed several additional experiments. First, western blot analysis of DARTS samples confirmed our results and revealed the stability of HK to SLC3A2 at the tested concentrations (fig. 3E). In addition, target engagement of HK was studied using the cellular thermal displacement assay (CETSA). SLC3A2 showed temperature-dependent instability, mainly precipitating at 55 deg.C, while HK (25 μ M) prevented precipitation of SLC3A2 even at 70 deg.C (FIG. 3F). Therefore, we subsequently compared the effect of different concentrations of HK in the CETSA assay at 55 ℃. The results show that HK can increase the stability of SLC3A2 at 55 ℃ concentration-dependently (fig. 3G). To further validate our findings, we performed DARTS and in vitro TSA with purified SLC3A2 protein. Consistent with the DARTS and CETSA results for cell lysates, HK apparently stabilized purified SLC3A2 (FIGS. 3H-3J).
All of the above results demonstrate that HK interacts directly with SLC3A2 in THP-1 cells.
Experimental example 4 SLC3A2 silencing inhibits NLRP3 inflammasome activation
Since we have demonstrated that HK interacts directly with SLC3A2, we subsequently explored whether SLC3A2 is associated with NLRP3 inflammasome activation. And further constructing a lentiviral shRNA expression vector to silence the expression of the SLC3A2 in the THP-1 cell. We designed three shRNA sequences to inhibit expression of SLC3A2, with sh-SLC3A2 # having the best silencing effect, and used for further studies (figure 4A). IL-1 β secretion was significantly reduced in NLRP 3-activated sh-SLC3A2 THP-1 cells compared to NLRP 3-activated sh-NT THP-1 cells, and even lower IL-1 β secretion after HK treatment (FIG. 4B). Meanwhile, the deletion of SLC3A2 inhibits the cleavage of caspase-1 and the release of IL-1 beta in sh-SLC3A2 THP-1 cells, and the deletion of SLC3A2 significantly inhibits the oligomerization of ASC (FIG. 4C, 4D).
The above results demonstrate that SLC3A2 is an upstream signal for regulating the activation of NLRP3 inflammasome, and that inhibition of SLC3A2 inhibits the activation of NLRP3 inflammasome, i.e. HK shows the potential to inhibit the activation of NLRP3 inflammasome by interacting with SLC3A2.
Experimental example 5, HK promotes the degradation of SLC3A2 through proteasome pathway
We further investigated the effect of HK on SLC3A2. Previous proteomic analysis showed that SLC3A2 was lower in the HK treated group than in the LPS/ATP treated group (fig. 3C). The same results were obtained with the western blot assay (FIG. 5A). However, HK did not alter the mRNA level of the SLC3A2 gene in THP-1 cells (FIG. 5B), suggesting that HK might reduce its mRNA level by enhancing the post-translational modification of SLC3A2. Since both the autophagy and proteasome pathways are involved in protein degradation, we subsequently tested which pathway led to the degradation of SLC3A2. Our findings indicate that proteasome inhibitor MG-132, but not autophagy inhibitor 3-MA, inhibited the HK-induced reduction of SLC3A2 (fig. 5C), and immunofluorescence experiments also showed that MG132 can prevent the HK-induced reduction of SLC3A2 levels (fig. 5D), indicating that HK triggers SLC3A2 degradation by proteasome-mediated proteolysis. To further validate whether HK enhances the degradation of SLC3A2 by the ubiquitin/proteasome pathway, we treated THP-1 cells with ubiquitin E1 inhibitor PYR 41. HK significantly inhibited SLC3A2 degradation, while PYR41 blocked this effect (fig. 5E).
In conclusion, HK promotes ubiquitination proteolysis of SLC3A2 protein in a proteasome-dependent manner, thereby promoting degradation of SLC3A2, i.e. HK is an SLC3A2 inhibitor.
Experimental example 6, HK exerts NLRP3 inhibitory effect by inhibiting mTORC1 activity
SLC3A2 is a single transmembrane glycoprotein that can interact with SLC7A5 through disulfide bonds as a chaperone, transporting large amounts of neutral essential amino acids such as leucine, valine, isoleucine, etc. Then, HK versus N was investigated 15 -the effect of leucine transport. The results show that HK significantly reduced the leucine content entering cells and lysosomes (fig. 6a,6 b). Confocal analysis of SLC3A2 showed that HK promoted degradation of SLC3A2, and co-localization of SLC3A2 with the lysosomal marker protein LAMP2 showed that HK reduced recruitment of SLC3A2 to the lysosomal surface (fig. 6C), which explains the reduction of leucine in cells and lysosomes.
The amino acid leucine is a potent stimulator of mTORC1, which plays an important role in NLRP3 inflammasome activation through hexokinase 1. To confirm whether HK inhibits activation of NLRP3 inflammasome by the SLC3 A2-regulated mTORC1 pathway, we investigated the effect of HK on mTORC1 by analyzing p70S6K1 total and phosphorylated protein levels. It was found that HK treatment did inhibit the phosphorylation level of p70S6K1 in LPS and LPS/ATP stimulated cells (FIG. 6D, 6E). Compared to LPS/ATP-treated cells, addition of mTOR agonist MHY1485 slightly activated mTORC1, but no statistically significant difference was observed (fig. 6d,6 e). Upon simultaneous treatment of THP-1 cells with HK, IL-1 β secretion was significantly reduced, while HK and MHY1485 treatment increased IL-1 β secretion slightly (FIG. 6F). The above results indicate that HK inhibits the activity of mTORC 1. To determine the mechanism by which HK-dependent mTORC1 modulation correlates with inflammatory-corpuscle restriction, NLRP3 inflammatory-corpuscle activation assays were performed using rapamycin. Consistent with previous reports, rapamycin may inhibit activation of NLRP3 inflammasome (fig. 6G).
These results indicate that HK exerts NLRP3 inflammasome inhibitory activity by inhibiting mTORC1 activity.
Experimental example 7 HK treatment improves survival rate of mouse septicemia and inhibits DSS-induced ulcerative colitis
The in vivo anti-inflammatory activity of HK was preliminarily examined in a mouse model of sepsis. HK significantly reduced serum IL-1 β compared to LPS group (FIG. 7A), but had no effect on TNF- α (FIG. 7B). At the same time, HK also greatly improved the survival of lethal endotoxic shock mice (fig. 7C). Based on our data from mouse sepsis models, HK might be suitable for the treatment of acute inflammatory diseases.
DSS is considered to be the most common inducer of colitis because it increases colonic permeability due to its toxicity to epithelial cells. Previous studies demonstrated that DSS-induced colitis is an experimental model of inflammation mediated by NLRP3 inflammatories. We then further investigated the anti-inflammatory activity of HK in vivo using DSS-induced acute ulcerative colitis. As a result, DSS was found to cause acute colitis in C57BL/6 mice, manifested by weight loss, diarrhea and pronounced rectal bleeding. Oral administration of HK (70 mg/kg) significantly reduced body weight loss in mice, whereas HK (35 mg/kg) was less effective on day 10 (FIG. 7D). To quantify the severity of colitis, a Disease Activity Index (DAI) score was determined. HK dose-dependently reduced DSS-induced DAI in mice (fig. 7E) and significantly attenuated colon length shortening (fig. 7F). Histological evaluation showed crypt distortion, goblet cell loss and inflammatory cell infiltration in colon tissue of colitis mice, while HK (70 mg/kg) improved these conditions (fig. 7G). Our studies also showed that SLC3A2 expression was highly upregulated in colon tissue of DSS-induced colitis mice. Colonic expression of SLC3A2 and IL-1 β was significantly increased in DSS group compared to control group (fig. 7h,7i).
In summary, the present inventors found in the study that honokiol targets SLC3A2 to inhibit the activation of NLRP3 inflammasome, and found by the mechanism of NLRP3 inhibitory activity on honokiol: honokiol can act directly on SLC3A2 protein. Further, it is found that honokiol can promote the ubiquitination degradation of SLC3A2 protein by proteasome, which results in the decrease of the content of leucine entering the cell, and the decrease of the content of SLC3A2 protein recruited to the surface of lysosome after degradation, thereby reducing the amount of leucine entering the lysosome, inhibiting the activity of mTORC1 and exerting NLRP3 inhibitory activity. In addition, in a DSS-induced colitis model, SLC3A2 is highly expressed, and the expression of SLC3A2 is reduced after HK treatment, which indicates that SLC3A2 can be used as a potential therapeutic target for NLRP3 inflammatory corpuscle abnormal activation related diseases. The invention considers that SLC3A2 is targeted by adopting a direct or indirect method, so that the content of SLC3A2 is reduced, an NLRP3 inflammatory body can be inhibited, and SLC3A2 can be used as a potential therapeutic target of diseases related to abnormal activation of the NLRP3 inflammatory body.
In conclusion, the invention proves that honokiol can be used as an SLC3A2 inhibitor for the first time, and the proteasome dependent degradation of honokiol is enhanced through direct interaction with SLC3A 2; the SLC3A2 inhibitor can influence the transportation of leucine in THP-1 cells and inhibit the activation of mTORC1, so that the activation of NLRP3 inflammasome is inhibited, the anti-inflammatory effect is realized, the obvious treatment effect is realized on acute inflammatory diseases such as septicemia and ulcerative colitis, and a new way is provided for the screening and research of anti-inflammatory drugs.
Claims (10)
- Use of an SLC3A2 inhibitor in the manufacture of an anti-inflammatory medicament.
- 2. The use of claim 1, wherein said SLC3A2 inhibitor is an agent that decreases the level of SLC3A2 in the body.
- 3. Use according to claim 2, wherein the SLC3A2 inhibitor is an agent that inhibits the expression of the SLC3A2 gene or an agent that promotes the degradation of the SLC3A2 protein, preferably an agent that promotes the degradation of the SLC3A2 protein.
- 4. The use of claim 3, wherein the SLC3A2 inhibitor is an agent that attenuates the ability of a cell to take up leucine.
- 5. The use of claim 4, wherein the SLC3A2 inhibitor is an agent that inhibits the activity of mTORC 1.
- 6. The use of claim 5, wherein the SLC3A2 inhibitor is an agent that inhibits NLRP3 activity.
- 7. The use of claim 1, wherein the anti-inflammatory agent is an agent for the treatment of an acute inflammatory disease.
- 8. The use according to claim 7, wherein the medicament is a medicament for the treatment of sepsis and/or ulcerative colitis.
- 9. Use of honokiol as an SLC3A2 inhibitor.
- 10. Use according to claim 9, wherein the SLC3A2 inhibitor is an agent which reduces the SLC3A2 level in the body or an agent which inhibits the activity of SLC3A2, preferably an agent which acts directly on the SLC3A2 protein to reduce the SLC3A2 level in the body.
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Non-Patent Citations (4)
| Title |
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| LI N等: "Effects of Honokiol on Sepsis-Induced Acute Kidney Injury in an Experimental Model of Sepsis in Rats", 《INFLAMMATION》, vol. 37, no. 4, pages 1191 - 1199 * |
| NAN WANG等: "Honokiol alleviates ulcerative colitis by targeting PPAR-γ–TLR4–NF-κB signaling and suppressing gasdermin-D-mediated pyroptosis in vivo and in vitro", 《INTERNATIONAL IMMUNOPHARMACOLOGY》, vol. 111, pages 1 - 13 * |
| TE I. WENG等: "Honokiol rescues sepsis-associated acute lung injury and lethality via the inhibition of oxidative stress and inflammation", 《INTENSIVE CARE MED》, vol. 37, pages 533, XP019884392, DOI: 10.1007/s00134-010-2104-1 * |
| 翟蒙恩等: "和厚朴酚对小鼠脓毒症心肌损伤的保护作用", 《西北大学学报( 自然科学版) 》, vol. 48, no. 6, pages 787 - 792 * |
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