Application of eucommia ulmoides lignan in preparation of medicine for preventing and treating sepsis

Abstract

The invention discloses application of Eucommia lignan in preparation of drugs for preventing and treating sepsis in the technical field of medicines. According to the scheme, the effects of the eucommia ulmoides lignan on the aspects of relieving sepsis inflammatory response, regulating cell phenotype and related protein gene expression, relieving neuron damage and the like are fully verified from the cell and animal levels, and the application value of the eucommia ulmoides lignan in preparation of the medicine for preventing and treating sepsis is proved.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of eucommia ulmoides lignans in preparing drugs for preventing and treating sepsis. Background Art

[0002] Eucommia ulmoides is an ancient tree species left over from the glacial movement of the third century. It is a second-level rare protected plant in my country. It has the effects of lowering blood pressure, lowering blood sugar, anti-tumor, antibacterial, antiviral, anti-inflammatory, antioxidant, anti-osteoporosis, and protecting the liver and kidneys. The chemical components extracted from Eucommia ulmoides can be divided into lignans, iridoids, flavonoids, phenylpropanoids, phenols, sterols, triterpenes, polysaccharides, etc. according to their structure. Among them, lignans are the most abundant and most studied active substances in Eucommia ulmoides. Studies have shown that Eucommia ulmoides lignans have the effects of lowering blood pressure, inhibiting microvascular damage caused by diabetes, and inhibiting neurodegeneration caused by glaucoma, and have neuroprotective effects.

[0003] Sepsis brain injury is part of the multiple organ dysfunction caused by sepsis. More than half of sepsis patients show symptoms of septic encephalopathy, and it may be the primary manifestation of sepsis patients before admission to the hospital. Septic encephalopathy can increase the mortality rate of sepsis patients and increase the risk of survivors leaving long-term neurocognitive deficits and mental disorders and other sequelae. At least 20% of patients who recover from sepsis have some degree of cognitive and emotional disorders and other sequelae, which seriously affect normal work and life and bring a heavy burden to patients' families and society.

[0004] The pathological mechanism of sepsis-induced brain injury is very complex. Neuroinflammation is an important pathological process of sepsis. Endothelial cells are activated, leading to changes in the blood-brain barrier, the production of neurotoxic factors, and the activation of microglia, which leads to oxidative stress, brain dysfunction, and neuronal apoptosis. In sepsis, the proinflammatory factors IL-1β and TNF-α in peripheral blood and brain are increased. The increased expression of TNF-α promotes the expression of cell adhesion molecules, chemokines, etc., induces the depolymerization of actin and the generation of intercellular spaces between endothelial cell skeletons, leading to blood-brain barrier dysfunction. The blood-brain barrier provides a large interface for cytokines to affect central nervous system function. The action of cytokines will destroy the function of the blood-brain barrier, resulting in increased permeability of the blood-brain barrier, which is more conducive to the entry of harmful substances into brain tissue and aggravates brain damage.

[0005] There is currently a lack of effective therapeutic drugs, and research on therapeutic drugs targeting its pathogenesis is an urgent issue to be addressed. Summary of the invention

[0006] The present invention aims to provide the use of eucommia ulmoides lignans in the preparation of drugs for preventing and treating sepsis, so as to inhibit the neurological sequelae caused by sepsis brain damage.

[0007] The present invention relates to the application of the eucommia ulmoides lignans in the preparation of drugs for preventing and treating sepsis.

[0008] Furthermore, the eucommia ulmoides lignans are eucommia ulmoides lignans extracts.

[0009] Furthermore, the eucommia ulmoides lignans can be used as an active substance in the form of an extract to prepare a pharmaceutical preparation. The eucommia ulmoides lignans of the present invention can be processed and prepared into pharmaceutical preparations in any unit dosage form suitable for taking, and these pharmaceutical preparations can be in the following dosage forms: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, oral liquids, lozenges, granules, granules, pills, powders, pastes, pills, suspensions, powders, solutions, suppositories, ointments, plasters, creams, sprays, drops or patches; when necessary, they can be made into dilute preparations and enteric preparations.

[0010] The working principle and beneficial effects of this scheme: The present invention clarifies for the first time that eucommia lignans have significant application value in the preparation of drugs for the prevention and treatment of sepsis. Through cell experiments and animal experiments, it has been confirmed that eucommia lignans can reduce the inflammatory response of septic cells and animal models, promote microglial phenotypic transformation, regulate related protein and gene expression, and reduce neuronal damage. This provides a new direction and material basis for the development of new sepsis treatment drugs, which is expected to bring better therapeutic effects to sepsis patients, reduce mortality, and reduce neurological sequelae, and has important clinical significance and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 ELISA was used to determine the effect of Eucommia ulmoides lignans on the inflammatory factors released by N9 and HT22 cells induced by LPS;

[0012] Figure 2 Immunofluorescence was used to detect the effect of Eucommia ulmoides lignans on the phenotype of N9 cells induced by LPS;

[0013] Figure 3 Flow cytometry was used to detect the effect of eucommia ulmoides lignans on the phenotype of N9 cells induced by LPS;

[0014] Figure 4 To detect the effect of Eucommia ulmoides lignans on the entry of Nrf2 protein into the cell nucleus by immunofluorescence;

[0015] Figure 5 Eucommia ulmoides lignans reduce the levels of plasma inflammatory factors in septic mice;

[0016] Figure 6 Eucommia ulmoides lignans alleviate neuronal damage in CLP mice;

[0017] Figure 7 The effect of Eucommia ulmoides lignans on the mRNA expression of target genes in CLP mice;

[0018] Figure 8 Effects of Eucommia ulmoides lignans on the hippocampus of CLP mice. DETAILED DESCRIPTION

[0019] The following is a further detailed description through specific implementation methods:

[0020] The preparation method of eucommia ulmoides lignans is extracted by referring to the preparation method of Example 1 in the Chinese patent "Application of eucommia ulmoides lignans in preparing drugs for preventing and treating hypertensive renal damage" with application number "CN201110169896.2".

[0021] Example

[0022] Preparation method of eucommia ulmoides lignans

[0023] S1: Grind 1 kg of dried Eucommia bark into 200 mesh to obtain Eucommia bark powder;

[0024] S2: The Eucommia bark powder obtained in S1 was suspended in a two-phase solvent of 3 kg anisole and 6 kg water, and heated to 60°C for extraction for 1 h;

[0025] S3: After the extraction in S2 is completed, the filtrate is filtered, and the filtrate is allowed to stand for stratification to separate the anisole phase; ammonium sulfate is added to the aqueous phase until saturated, and then 2 kg of ethanol is added for extraction, and the ethanol phase is recovered;

[0026] S4: The anisole phase and the ethanol phase are combined, the solvent is removed under reduced pressure, and the residue is separated using a macroporous resin, and the elution process is 2 column volumes of water, 2 column volumes of 20% ethanol, 3 column volumes of 50% ethanol, and 2 column volumes of 60% ethanol. The 50% ethanol and 60% ethanol eluates are combined, concentrated, and dried to obtain the lignan extract. The total lignin content in the extract is detected to be 57.3%.

[0027] Experiment 1: Cell experiment

[0028] Cell culture test materials

[0029]

[0030] Cell culture: HT22 cells (CL-0595, Wuhan Pronocell) were cultured in high-glucose DMEM complete medium (KGM31800S-500, Keygen Biotechnology), and N9 cells (C0927, Shanghai Guandao Biotechnology) were cultured in R1640 (2192673, Gibco) medium supplemented with Gibco serum (10099-141, Gibco) and L-glutamine (G0200, Solebol).

[0031] Establishment of inflammation model and drug treatment: Cell suspension (400,000 / well) was inoculated in a 6-well plate, and after pre-culture for 12 hours, 1 μg / ml LPS was added to stimulate for 24 hours to establish the inflammation model. The Eucommia ulmoides lignans group was pre-incubated with 100 μg / ml Eucommia ulmoides lignans 2 hours before LPS.

[0032] Detection indicators

[0033] ELISA was used to determine the expression level of inflammatory factors: Mouse tumor necrosis factor α (TNF-α) kit (Cat. No. 430904, Biolegend), mouse interleukin 6 (IL-6) kit (Cat. No. 431304, Biolegend), and mouse interleukin IL-1β (IL-1β) kit (Cat. No. 432604, Biolegend) were used according to the instructions. Within 15 minutes after termination, the absorbance (OD value) of each well at a wavelength of 450 nm was measured using an automatic microplate reader (WD-2102B, Liuyi) to detect the expression of TNF-α, IL-6, and IL1β in the cell supernatant. The results showed that Eucommia ulmoides lignans can reduce LPS-induced cellular inflammation. Figure 1 .

[0034] Western Blot was used to determine the expression of target proteins: the expression of target genes such as AR, Nrf2, AMPK, p-AMPK, BECLIN-1, and LC3 in cells was determined. The primary antibodies used included Rabbit Anti AR (DF6554, Affinity, 1 / 1000), etc., and the secondary antibodies were horseradish enzyme-labeled goat anti-rabbit IgG (H+L) (ZB-2301, Zhongshan Jinqiao, 1 / 2000), etc. The results showed that Eucommia ulmoides lignans could reduce the expression of AR, Beclin1, and LC3II / LC3I in N9 cells and HT22 cells induced by LPS, increase the expression inhibition of AMPK and p-AMPK caused by LPS, and reverse the inhibitory effect of LPS on Nrf2 expression. Figure 2 .

[0035] Flow cytometry was used to detect the M1 and M2 phenotypes of N9 cells: cells were collected and stained and then detected by flow cytometry. The results showed that Eucommia ulmoides lignans could promote the transformation of N9 microglia from the pro-inflammatory M1 type to the anti-inflammatory M2 type. Figure 3 .

[0036] Immunofluorescence: A cover glass was placed in a 6-well plate, and the cell suspension was inoculated. After pre-culture, LPS stimulation and eucommia lignans treatment were performed. After the staining step, the cells were fixed with mounting media and detected by laser confocal microscopy (FV1000, Olympus, JAPAN). The results showed that LPS stimulation increased the nuclear translocation of Nrf2, and eucommia lignans promoted the nuclear translocation of Nrf2. Figure 4 .

[0037] Experiment 2: Animal Experiment

[0038] Establishment of mouse CLP model: 8-week-old C57BL / 6 male mice were used and divided into 5 groups, each with 12 mice, namely sham group (Sham), CLP model group (CLP), low-dose Eucommia lignans + CLP group (300 mg / kg / d), high-dose Eucommia lignans + CLP group (450 mg / kg / d), and epalrestat + CLP group (100 mg / kg / d). The animals were fasted at 9 am on the day of surgery, and the surgical model was established in the afternoon. Using isoflurane respiratory anesthesia, the skin was prepared, and the mice were fixed in the supine position. A vertical incision of about 1 cm long was made along the linea alba at 1-1.5 cm below the xiphoid process, the cecum was freed, ligated in the middle of the cecum, and punctured twice at the lower end of the cecum with a 0.8 mm diameter needle. After squeezing out the feces, the cecum was summarized and sutured layer by layer. After suturing, 1 ml of 0.9% sodium chloride injection was injected subcutaneously for fluid replacement, and the mice were placed in a warming pad for resuscitation. After modeling, normal saline was injected subcutaneously every morning, and 5% glucose injection was injected subcutaneously in the evening. The eucommia lignans group and the epalrestat group were given drugs by gavage, and the other groups were given solvents by gavage. The drugs were given once a day 4 days before surgery. The modeling was done on the 5th day. After surgery, the drugs were given once a day for a total of 3 days. The gavage volume was 100μl / 10g.

[0039] Detection indicators

[0040] ELISA detection of plasma inflammatory factors: Blood was collected 24 hours after modeling, and ELISA was used to detect the indicators of inflammatory factors in hippocampal plasma. The results showed that eucommia ulmoides lignans and epalrestat could reduce the levels of TNF-α and IL6 in the plasma of CLP mice. Figure 5 .

[0041] HE detection of hippocampal pathological morphology: The hippocampal tissue was taken for HE staining to observe the pathological morphology. The results showed that the hippocampal structure of the brain tissue in the sham operation group was clear, and the neuronal cells were not damaged; in the CLP group, obvious neuronal cell damage was observed in the DG region of the hippocampus, with condensed and darkly stained nuclei; all drug-treated groups had a certain therapeutic effect, among which the epalrestat group had the best effect, the low-dose Eucommia ulmoides lignans group had fewer damaged neuronal cells, and the high-dose Eucommia ulmoides lignans group had significantly improved neuronal cell damage. Figure 6 .

[0042] q-PCR and Western Blot detection of gene and protein expression: Detect the expression of each target gene at the mRNA and protein levels. The q-PCR results showed that compared with the sham operation group, the mRNA levels of AR, Beclin1, LB3A, and LC3B in the CLP model group increased, the expression of Nrf2 decreased, and there was no significant change in AMPK-a2; compared with the CLP model group, the expression of LC3A and LC3B decreased after low-dose and high-dose eucommia ulmoides lignans treatment, and the expression of Nrf2 increased. Figure 7 .

[0043] Immunofluorescence double staining: Detect changes in the phenotype of hippocampal microglia. The results showed that compared with the sham operation group, the expression of CD68 in the CLP model group was upregulated, and the expression of CD68 was reduced after treatment with low and high doses of Eucommia ulmoides lignans and epalrestat. Figure 8 .

[0044] In summary, Experiment 1 and Experiment 2 fully verified the role of Eucommia ulmoides lignans in preventing and treating sepsis at the cellular and animal levels, respectively. In cell experiments, Eucommia ulmoides lignans can significantly reduce inflammatory responses, regulate microglial phenotypes, and reduce the expression of related proinflammatory proteins and genes; in mouse inflammation model experiments, Eucommia ulmoides lignans can not only reduce inflammatory responses, but also reduce CLP-induced mouse hippocampal neuronal cell damage. Its mechanism of action may be to promote Nrf2 nuclear translocation, thereby enhancing antioxidant capacity and inhibiting the activity of proinflammatory M1 microglia, proving that it has important application value in the preparation of drugs for the prevention and treatment of sepsis.

[0045] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. Application of Eucommia ulmoides lignans in the preparation of drugs for the prevention and treatment of sepsis.

2. The use of the eucommia ulmoides lignans according to claim 1 in the preparation of a drug for preventing and treating sepsis, characterized in that: The eucommia ulmoides lignans are eucommia ulmoides lignans extracts.

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