CN113975275B - New use of piperlongumine for inhibiting programmed necrosis - Google Patents

New use of piperlongumine for inhibiting programmed necrosis Download PDF

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CN113975275B
CN113975275B CN202111429558.8A CN202111429558A CN113975275B CN 113975275 B CN113975275 B CN 113975275B CN 202111429558 A CN202111429558 A CN 202111429558A CN 113975275 B CN113975275 B CN 113975275B
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查运红
何小燕
李敏
魏钧
方明
胡昕倩
余雅婕
叶芷
赵海龙
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Yichang No1 People's Hospital (people's Hospital Of China Three Gorges University)
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Abstract

The invention belongs to the technical field of medicines, and discloses a natural product, namely Piperlongumine (Piperlongumine), which can effectively inhibit programmed cell necrosis of Receptor-interacting protein kinase-1 (Receptor-interacting protein kinase 1, RIPK1) dependence in vivo and in vitro and has low toxicity. The present invention addresses the existing therapeutic bottlenecks and provides new protocols and hopes for the treatment of such related diseases characterized by aberrant activation of programmed necrosis.

Description

New use of piperlongumine for inhibiting programmed necrosis
Technical Field
The invention relates to application of piperlongumine as a programmed necrosis inhibitor, and belongs to the technical field of medicines.
Background
Programmed Cell Death (PCD) has been newly discovered in recent decades, and following stimulation with Tumor necrosis factor α (TNF α), RIPK1 (receptor interacting protein kinase 1), RIPK3 (receptor interacting protein kinase 3), and MLKL (mixed lineage kinase domain-like pseudokinase) can form "programmed necrosis" (neosome) and induce neotosisis, which can be rescued by key regulatory molecule RIPK1 kinase specific inhibitor, necostatin-1 s (Nec-1 s). Linde et al found that inhibitors of programmed necrosis protected hypothermia and extended survival in a mouse model of SIRS induced by tail vein injection of TNF- α.
The active death of cells is indispensable to the physiological activities of normal development and aging of the organism, resistance to invasion of pathogenic microorganisms, maintenance of homeostasis and the like, and the imbalance of the cells often causes various diseases such as developmental deformity, immune system diseases, neurodegenerative diseases, cancer and the like, and even death of individuals. Therefore, the intervention on the programmed cell death has important significance for the research of disease treatment.
RIPK1 and RIPK3 are involved in inflammation, cellular stress and physiological responses following infection. Environmental cues (e.g., bacterial or viral infection, pro-inflammatory stimuli, or cellular stress) activate these pathways, producing complex signals that play a central role in RIPK1, which determines cell fate. Many inflammatory, infectious and degenerative diseases are driven by RIPK1 kinase activity, which makes it a valuable target for disease treatment.
The reviews published in 7/15.2020 further summarize human diseases related to RIPK1, including inflammatory bowel disease (crohn's disease), multiple sclerosis, and systemic lupus erythematosus, which are mediated by RIPK 1. There have been several studies and clinical data from different research groups that suggest that amyotrophic lateral sclerosis, ischemia reperfusion injury, ischemic stroke, hemorrhagic stroke, frontotemporal dementia, alzheimer's disease, huntington's disease, duchenne's muscular dystrophy are closely associated with RIPK1 activation and programmed necrosis.
Therefore, the research finds that the inhibitor of the cell apoptosis has very important significance for treating the diseases related to the cell death.
Piperlongumine (CAS #: 20069-09-4), piperlongumine (piplartine), piper Long Ming are amide alkaloid compounds extracted from common traditional Chinese medicine materials with homology of medicine and food and Piper longum of Piperaceae, are cultivated in Guangdong, guangxi, yunnan and the like in China, and have very rich resources.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of piperlongumine as a programmed necrosis inhibitor.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the application of piperlongumine as an RIPK1 inhibitor.
Further preferably, the piperlongumine is applied to inhibiting T/S/Z-induced programmed necrosis on human colon cancer cells HT-29, human T lymphocyte leukemia cell strains (FADD defficient Jurkat) and human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM).
Further preferably, the piperlongumine is used as the RIPK1 inhibitor for treating neurodegenerative diseases: amyotrophic lateral sclerosis, frontotemporal dementia, motor neuron disease, multiple sclerosis, alzheimer disease, huntington disease, spinocerebellar ataxia, becker muscular dystrophy, cerebral hemorrhage, ischemia reperfusion injury (ischemic stroke, neonatal brain injury, myocardial ischemia, renal ischemia reperfusion injury, retinal ischemia, brain trauma), crohn's disease, acute pancreatitis, organ transplantation, liver disease (hepatitis, hepatocarcinoma), age-related macular degeneration, neocoronary pneumonia, systemic lupus erythematosus, psoriasis, systemic inflammatory response syndrome, etc.
The invention has the following beneficial effects:
(1) The piperlongumine can effectively inhibit RIPK 1-dependent apoptosis in vitro and has low toxicity.
(2) Piperlongumine can be used as a programmed necrosis inhibitor for treating neurodegenerative diseases: amyotrophic lateral sclerosis, frontotemporal dementia, motor neuron disease, multiple sclerosis, alzheimer's disease, huntington's disease, spinocerebellar ataxia, becker muscular dystrophy, cerebral hemorrhage, ischemia reperfusion injury (ischemic stroke, neonatal brain injury, myocardial ischemia, renal ischemia reperfusion injury, retinal ischemia, brain trauma), crohn's disease, acute pancreatitis, organ transplantation, liver disease (hepatitis, liver cancer), age-related macular degeneration, neocoronary pneumonia, systemic lupus erythematosus, psoriasis, systemic inflammatory response syndrome, and other related diseases.
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FIG. 1 protection of different cell lines against programmed necrosis by Piperlongumine (Piperlongumine);
figure 2 Piperlongumine (Piperlongumine) rescues IC50 and EC50 of programmed necrosis in different cell lines: A. treating HT-29 cells with varying concentrations of Piperlongumine (Piperlongumine); B. treating FADD-/-Jurkat cells with varying concentrations of piperlongumine; C. treating CCRF-CEM cells with varying concentrations of piperlongumine; D. in HT-29 cells, cells were pretreated with varying concentrations of Piperlongumine +200nM SM-164+20 μ M zVAD; E. in FADD-/-Jurkat cells, cells were pretreated with varying concentrations of piperlongumine +200nM SM-164+20 μ M zVAD; F. in CCRF-CEM cells, cells were pretreated with varying concentrations of piperlongumine +200nM SM-164+20 μ M zVAD.
FIG. 3 Effect of Piperlongumine (Piperlongumine) on markers of programmed necrosis: phosphorylation at the S321 site of RIPK1 kinase;
FIG. 4 the survival curves of mice with TNFa-induced systemic inflammatory response syndrome and anus Wen Futu protected by piperlongumine.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
This example discloses the use of piperlongumine as an inhibitor of apoptosis.
Preferably, the piperlongumine is effective in inhibiting RIPK1 dependent apoptosis.
Further preferably, the application of the piperlongumine in inhibiting T/S/Z induced programmed necrosis is provided.
Further preferably, the use of piperlongumine for inhibiting T/S/Z induced apoptosis in human cells.
In this example, 20ng/ml hTNF-alpha is added to induce apoptosis necrosis of cells after the cells are pretreated for 30min with DMSO, 200nM SM-164+20 μ M zVAD (S/Z), 200nM SM-164+20 μ M zVAD +25 μ M Nec-1 (S/Z/N), 200nM SM-164+20 μ M zVAD +10uM Piperlongumine (Piperlongumine) (S/Z/PL) in human colon cancer cells (HT-29), human T lymphocyte leukemia cell strains (FADD defficient Jurkat, FADD-/-Jurkat) and human acute lymphoblastic leukemia T lymphocytes (CCRF-CEM), and ATP assay is used to detect the number of surviving cells.
Example 1 Piperlongumine inhibits apoptosis
As shown in FIG. 1, piperlongumine (PL) inhibited the occurrence of T/S/Z (TNF-. Alpha./SM-164/zVAD) -induced apoptosis, and the specific results are shown in Table 1.
TABLE 1 different cell lines, rescue of apoptosis results with Piperlongumine
Figure BDA0003379632200000041
Note: t: TNF alpha; s: SM164; z: zVAD; n: nec-1 (as a positive control for apoptosis inhibitor); PL: piperlongumine. In human cell lines, the piperlongumine group was compared to the model group, (. X.p < 0.0001).
Example 2: the safety and effectiveness of the piperlongumine in different cell lines.
HT-29 cells, FADD-/-Jurkat cells, and CCRF-CEM cells were treated with varying concentrations of Piperlongumine (Piperlongumine), and the number of viable cells was determined using ATP assay (see FIGS. 2A-C).
On the basis, after cells were pretreated for 30min by adding 200nM SM-164+20 μ M zVAD, programmed necrosis was induced by 20ng/ml hTNF- α, and the number of surviving cells was examined by ATP assay (see FIGS. 2D-F).
Specific results are shown in FIG. 2, and tables 2 and 3, the EC50 values of Piperlongumine (Piperlongumine) in HT-29, FADD-/-Jurkat and CCRF-CEM cells are 0.484uM, 2.403uM and 11.34uM, respectively, and the toxicity is low (HT-29IC50. Experimental results also show that 5uM-10uM of piperlongumine can obviously inhibit apoptosis induced by human tumor necrosis factor (human TNFa, hTNF-alpha) and improve the survival rate of cells.
TABLE 2 comparison of cell viability after Piperlongumine treatment at different concentrations
Figure BDA0003379632200000042
Figure BDA0003379632200000051
TABLE 3 comparison of cellular activities of Piperlongumine at different concentrations to inhibit apoptosis
Figure BDA0003379632200000052
Note: all cells were treated with TNF α/SM164/zVAD to induce apoptosis.
Example 3 Piperlongumine (Piperlongumine) inhibits the expression of p-RIPK1
Treatment of HT-29 cell lines with T/S/Z and hTNFa treatment of FADD -/- The Jurkat cell line induces programmed necrosis of cells, and the addition of the piperlongumine can obviously save the programmed necrosis of the cells within 2 hours. Collecting cell samples at 0, 1 and 2 hours, extracting protein, performing western blotting, and inhibiting apoptosis activation marker p-RIPK1 (S166) to save cell death.
Example 4 Piperlongumine improves RIPK 1-dependent cell death
A method of injecting 2 mu g TNF alpha into the tail vein of an 8-week-old C57BL/6 mouse is adopted to successfully establish a SIRS mouse model. As shown in FIG. 4, the RIPK 1-dependent cell death pathway is involved in TNF-. Alpha.induction of Systemic Inflammatory Response Syndrome (SIRS). The piperlongumine can obviously improve the hypothermia state (P < 0.05) of the mice and improve the survival rate (P < 0.05) of the mice, and specific results are shown in tables 4 and 5.
FIG. 4 shows that Piperlongumine (Piperlongumine) can significantly improve the survival rate and body temperature of SIRS mice. The application of Piperlongumine (Piperlongumine) in a mouse model with abnormal activation of programmed necrosis as a characteristic systemic inflammatory response syndrome can obviously improve the hypothermia state of a mouse (P < 0.05) and improve the survival rate of the mouse (P < 0.05).
TABLE 4 comparison of survival counts for differently treated mice in mTNF- α induced SIRS mouse model
Figure BDA0003379632200000061
TABLE 5 comparison of body temperatures of differently treated mice in mTNF- α induced SIRS mouse model (Mean + -SD)
Figure BDA0003379632200000062
Figure BDA0003379632200000071
The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (3)

1. Use of piperlongumine for the manufacture of a medicament for the treatment of a disease associated with programmed necrosis, wherein the disease associated with programmed necrosis is systemic inflammatory response syndrome.
2. The use according to claim 1, wherein the systemic inflammatory response syndrome is TNF- α induced.
3. Use according to any of claims 1 or 2, wherein the medicament is capable of ameliorating a hypothermic condition.
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