Lung purging decoction drug serum with anti-neuritis effect

Abstract

The invention relates to the related technical field of pharmaceutical compositions, in particular to a Dabag lung-purging decoction drug serum with an anti-neuritis effect, the drug serum inhibits microglial cell neuroinflammation response by regulating an HIF-1 / VEGF / AA signal axis, and a preparation method of the drug serum comprises the following steps: taking 20-94 parts of fried semen lepidii, 20-94 parts of rheum officinale, 20-94 parts of radix paeoniae alba, 7-32 parts of honey-fried licorice root, 7-32 parts of scutellaria baicalensis and 7-32 parts of dried ginger; adding 8 times of water, soaking for 1 hour, and decocting twice; according to the Daixiang lung-purging decoction with the anti-neuritis effect, drug-containing serum and freeze-dried powder of 10% and 20% drug-containing serum of the Daixiang lung-purging decoction significantly increase the activity of neuroinflammatory cells, and the protein expression levels of TLR4, HIF-1alpha, i NOS, VEGF, COX-2 and ALOX5 in the neuroinflammatory cells are significantly reduced by cooperatively regulating key targets in an HIF-1 / VEGF / AA signal axis; the content of arachidonic acid (AA) is obviously increased, the content of inflammatory metabolites LTD4, PGE2 and TAX2 is reduced, and the cascade reaction of neuroinflammation is reduced; bax and Bc l-2 protein expression level are significantly down-regulated and up-regulated, so that nerve cell apoptosis is reduced, and the effect of protecting nerve cells is achieved.

Description

Technical Field

[0001] This invention relates to the field of drug combination technology, and in particular to a serum containing Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects. Background Technology

[0002] Microglia are the most important immune effector cells in the central nervous system (CNS), accounting for 10%-15% of the total number of glial cells in the brain, and belong to the mononuclear macrophage system. They are widely distributed in the brain and spinal cord, exhibiting high dynamism and functional diversity, and serve as the nervous system's "first line of defense." Studies have shown that microglia activation can trigger persistent neuritis, leading to neuronal death, and consequently promoting the occurrence and exacerbation of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, cerebral ischemia-reperfusion injury, or multiple sclerosis. Inhibiting glial cell activation can regulate neuroinflammatory responses and reduce pathological damage to brain tissue. Currently, commonly used anti-neuroinflammatory drugs suffer from problems such as single target action, significant side effects, and the potential for drug resistance with long-term use.

[0003] Traditional Chinese medicine (TCM) has demonstrated unique advantages in the regulation of neuroinflammation. Based on the TCM theory of "lung-brain correlation," lung qi stagnation can lead to phlegm and blood stasis disturbing the sensory orifices, which highly coincides with the "lung-brain axis" mechanism in modern medicine where lung inflammation induces central nervous system inflammation. Literature reports that the Yifei Xuanfei Jiangzhuo formula, guided by the theory of "treating from the lung," can improve neuroinflammation in vascular dementia by regulating microglial cell polarization. The Dunhuang ancient medical formula, Da Xie Fei Tang, originates from the Dunhuang manuscript *Fu Xing Jue Zang Fu Yong Yao Fa Yao*. Its prescription consists of six herbs: Lepidium seed, rhubarb, white peony root (3 liang each), licorice root (prepared), scutellaria root, and dried ginger (1 liang each). It is used for phlegm-heat stagnation in the lungs, and has the effects of purging the lungs, clearing phlegm, relieving cough, and calming asthma. Lepidium seed purges the lungs, calms asthma, promotes diuresis, and reduces swelling, helping to clear phlegm and turbidity from the body and reduce obstruction of the brain orifices. Rhubarb, processed with wine, clears the bowels, eliminates turbidity, and removes blood stasis; these two herbs are the principal ingredients. Scutellaria baicalensis clears heat, dries dampness, and drains fire; dried ginger warms the lungs and resolves phlegm; these two herbs are the assistant ingredients. White peony root astringes yin, stops sweating, nourishes blood, harmonizes qi and blood; this is the adjuvant ingredient. Licorice root relieves spasms, stops pain, and harmonizes the other herbs; this is the guiding ingredient. The treatment uses Scutellaria baicalensis to assist rhubarb in clearing away excess heat in the bowels; dried ginger assists Lepidium apetalum to warm and expel phlegm from the lungs; and roasted licorice root assists peony root, its sour and sweet properties nourishing yin and replenishing lung deficiency. The entire formula treats "lung excess syndrome, phlegm in the chest, shortness of breath preventing lying down, and also treats constipation, edema of the body and face, and unresolved phlegm." Modern pharmaceutical research shows that Da Xie Fei Tang can treat chronic obstructive pulmonary disease (COPD) and prevent pneumonia. Based on the "lung-brain correlation" theory, network pharmacology studies have shown that Da Xie Fei Tang can exert an anti-neuroinflammatory effect by regulating the HIF-1 / VEGF / AA signaling axis and simultaneously regulate lung and brain function. This "systemic intervention" model is more in line with the body's physiological characteristics. It can not only reduce the source of inflammation, block the spread of inflammation and optimize the repair environment, but also reduce the comorbidity risk of related diseases (such as neurodegenerative diseases and cerebrovascular diseases). Summary of the Invention

[0004] The purpose of this invention is to provide a serum containing Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects, which has a synergistic anti-neuritis effect through multiple pathways and multiple targets. To achieve the above objective, this invention provides the following technical solution: a serum containing Da Xie Fei Tang with anti-neuritis effects, wherein the serum inhibits the inflammatory response of microglia by regulating the HIF-1 / VEGF / AA signaling pathway, and its preparation method includes:

[0005] (1) Take 20-94 parts of stir-fried Lepidium apetalum, 20-94 parts of rhubarb, 20-94 parts of white peony root, 7-32 parts of roasted licorice root, 7-32 parts of Scutellaria baicalensis and 7-32 parts of dried ginger, soak them in 8 times the amount of water for 1 hour, and then decoct them twice.

[0006] (2) Combine the filtrates and concentrate them to a crude drug weight of 0.5-3 g / mL, then freeze-dry to obtain Da Xie Fei Tang freeze-dried powder;

[0007] (3) Dissolve the freeze-dried powder of Da Xie Fei Tang in 10-40 g / kg-1 SPF-grade Wistar rats were administered the dose by gavage for 5 minutes to 7 days.

[0008] (4) Blood was collected from the heart 5 min to 8 h after the last administration of the drug, and the serum was collected and freeze-dried to obtain the serum containing Da Xie Fei Tang.

[0009] Furthermore, the concentration of the drug-containing serum used is 5%-20% (v / v).

[0010] Furthermore, the anti-neuritis effect is manifested by LPS-induced decrease in BV2 cell viability and excessive secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α. Its anti-neuritis effect is confirmed by the following indicators:

[0011] (1) The viability of BV2 cells increased by 29% to 62%;

[0012] (2) Significantly reduced the mRNA expression levels and contents of pro-inflammatory factors IL-1β, IL-6, and TNF-α (P<0.01);

[0013] (3) Significantly increased the mRNA expression level and content of anti-inflammatory factors IL-4 and IL-10 (P<0.01);

[0014] (4) The content of pro-inflammatory mediator NO decreased by 7% to 15%.

[0015] Furthermore, the modulation of the HIF-1 / VEGF / AA signal axis is as follows:

[0016] The expression of TLR4, HIF-1α, VEGF, iNOS, ALOX5 and COX2 proteins was significantly downregulated (P<0.01);

[0017] The levels of LTD4, PGE2, and TXA2 in the ALOX5 and COX2-mediated arachidonic acid (AA) metabolic pathway decreased by 12%–27%.

[0018] The AA content increased by 4% to 22%.

[0019] Furthermore, the drug-containing serum simultaneously regulates apoptosis-related proteins:

[0020] (1) Bax protein expression decreased by 18% to 78%;

[0021] (2) Bc1-2 protein expression increased by 52% to 352%.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. Mechanism clearly defined: Lyophilized serum containing 10% and 20% Da Xie Fei Tang significantly increased the viability of neuroinflammatory cells. By synergistically regulating key targets in the HIF-1 / VEGF / AA signaling axis, it significantly downregulated the protein expression levels of TLR4, HIF-1α, iNOS, VEGF, COX-2, and ALOX5 in neuroinflammatory cells; significantly reduced the content of pro-inflammatory factor NO and the gene and protein expression levels of IL-6, IL-1β, and TNF-α, while increasing the gene and protein expression levels of anti-inflammatory factors IL-4 and IL-10; significantly increased the content of arachidonic acid (AA) and decreased the content of its inflammatory metabolites LTD4, PGE2, and TAX2, thereby reducing the neuroinflammatory cascade reaction; and significantly downregulated Bax and upregulated Bcl-2 protein expression levels, reducing neuronal apoptosis and exerting a protective effect on nerve cells.

[0024] 2. Clear pharmacodynamic material basis: GC-MS mass spectrometry has identified at least 53 effective compound components in the serum of the Da Xie Fei Tang of this invention, including amino acids, organic acids, sugars, saturated fatty acids, unsaturated fatty acids, and lipids. Attached Figure Description

[0025] Figure 1 This is a graph showing the effect of serum containing the Da Xie Fei Tang (a traditional Chinese medicine formula) of the present invention on LPS-induced BV2 cell viability and NO content.

[0026] Figure 2 The figure shows the effect of serum containing the Da Xie Fei Tang (a traditional Chinese medicine formula) of this invention on the levels of pro-inflammatory and anti-inflammatory factors induced by LPS in BV-2 microglia.

[0027] Figure 3 This is a graph showing the effect of serum containing the Da Xie Fei Tang (a traditional Chinese medicine formula) of the present invention on the levels of pro-inflammatory and anti-inflammatory factor mRNAs induced by LPS in BV-2 microglia.

[0028] Figure 4 This is a Western blot image showing the effect of serum containing the Da Xie Fei Tang decoction of the present invention on LPS-induced HIF-1 / VEGF / AA signaling axis and apoptosis protein production in BV-2 microglia;

[0029] Figure 5 This is a graph showing the effect of serum containing the Da Xie Fei Tang (a traditional Chinese medicine formula) of the present invention on the protein expression levels of LPS-induced HIF-1 / VEGF / AA signaling axis and apoptosis proteins in BV-2 microglia.

[0030] Figure 6 This is a diagram showing the effect of serum containing the Da Xie Fei Tang (a traditional Chinese medicine formula) of the present invention on the arachidonic acid metabolic pathway induced by LPS in BV-2 microglia.

[0031] Figure 7 This is the total ion chromatogram of serum containing Da Xie Fei Tang obtained by GC-MS detection in this invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The embodiments of the present invention are described below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where no specific technical or technical conditions are specified in the embodiments, they shall be carried out in accordance with the technical or technical conditions described in the literature in the field or in accordance with the product instructions. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be obtained commercially.

[0034] Materials and reagents used in the following examples:

[0035] 1. Cells: Mouse microglia (BV2) catalog number FH0355, purchased from Haixing Biotechnology Co., Ltd.

[0036] 2. Animal SPF grade: Wistar male rats, weighing 180-220g, provided by the Experimental Animal Center of Gansu University of Traditional Chinese Medicine, production license number: SCXK(Gan)2021-0001.

[0037] 3. Drugs and reagents

[0038] 3.1 The following herbs were purchased from Kuaikang Pharmacy in Lanzhou City, Gansu Province: Lepidium seed (batch number: 19012503), rhubarb (batch number: 23101809-1), white peony root (batch number: 231101), prepared licorice root (batch number: 230901), scutellaria root (batch number: 2203005), and dried ginger (batch number: 2211001).

[0039] 3.2 DMEM high-glucose medium, PBS (Hyclone, batch numbers: SH30022.01, SH300256.01); fetal bovine serum (TransSerum FQ, batch number: FS301-02); penicillin-streptomycin mixture (100X), trypsin-EDTA digestion solution, dimethyl sulfoxide (DMSO), lipopolysaccharide (Solarbio, P1400, T1300, 1121E0321, L8880); CCK-8 (Shangwei Biotechnology Co., Ltd., CK001-01); nitric oxide (NO) reagent kit (Solarbio, batch number: BC1470); BCA reagent kit, β-actin , AlOX5, HIF-1a, iNOS, TLR4, VEGF, Bcl-2, Bax, COX2 (Servicebio, G2026-200T, GB15003, GB111330, GB1 11339, GB115703, GB11519, GB11034B, GB154380, GB15690, GB115672); IL-1β, IL-6, TNF-α, IL-4, IL-10 ELISA kits (Thermo Fisher Scientific Co., Ltd., batch numbers: 416036-005, 403465-008, 418809-003, 390848-006, 393807-004); Arachidonic acid (AA), prostaglandin E2 (PGE2), thromboxane A2 (TXA2), leukotriene D4 (LTD4) (Jiangsu Enzyme Immunoassay Co., Ltd., batch numbers: MM-45088M1, MM-0062M1, MM-44461M1, MM-45661M1);

[0040] 3.3 Microplate reader (SpectraMax i3X, Molecular Devices); Inverted microscope (CKX41+DP21, OLYMPUS); CO2 incubator (CLM-170B-8-TC, Taicang Yisigao Medical Device Technology Co., Ltd.); Clean bench (BSC-1500ⅡA2-X, BIOBASE); 3D cryogenic grinder (KZ-5F-3D, Wuhan Saiweier Biotechnology Co., Ltd.); Real-time PCR instrument (CFX Connect, Bio-Rad, USA); Chemiluminescence analyzer (SCG-W3000, Wuhan Saiweier Biotechnology Co., Ltd.); Ultrasonic cell disruptor (JY92-11N); Vertical electrophoresis apparatus (Servicebio, SVE-2); Transfer electrophoresis apparatus (Servicebio, SVT-2).

[0041] Please see Figure 1-7This invention provides a technical solution: a serum containing Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects, which inhibits microglial neuroinflammatory responses by regulating the HIF-1 / VEGF / AA signaling axis. The preparation method includes:

[0042] Step 1: Preparation of Da Xie Fei Tang (a traditional Chinese medicine formula): Take 46g of stir-fried Lepidium apetalum, 46g of rhubarb, 46g of white peony root, 15g of prepared licorice root, 15g of Scutellaria baicalensis, and 15g of dried ginger, totaling 183g of medicinal materials. Add 8 times the amount of pure water and soak for 1 hour. After boiling over high heat, reduce to low heat and simmer for 1 hour. Filter the mixture. Add 6 times the amount of water to the dregs and continue to simmer for 40 minutes. Filter the mixture again. Combine the two filtrates and concentrate the filtrate to 183ml. Freeze-dry the mixture to obtain a 20% yield of freeze-dried Da Xie Fei Tang powder.

[0043] Step 2, Preparation of drug-containing serum: Male SPF-grade Wistar rats were randomly divided into three groups: blank (NC) group, Daxiefei Decoction (DXFT) group, and Piracetam (PIR) group, with 10 rats in each group. The Daxiefei Decoction group and the Piracetam group were each administered 40 g / kg of the drug by gavage. -1 and 0.324 g·kg -1 The control group was administered an equal volume of physiological saline by gavage. The drug was administered by gavage once a day for 7 consecutive days. Two hours after the last administration, blood was collected from the heart of each group of rats. After blood collection, the rats were allowed to stand for 30 minutes, centrifuged at 3000 r / min for 10 minutes, and the serum was inactivated in a water bath at 56℃ for 30 minutes. The serum was then filtered through a 0.22 μm filter for sterilization, dispensed, and freeze-dried to obtain the drug-containing serum freeze-dried powder of each group, which was stored at -20℃.

[0044] Step 3, Cell Culture: BV2 cells were seeded in DMEM complete medium containing a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin. The cells were then cultured in a 37°C, 5% CO2 incubator until they adhered to the wall and grew to about 80% and were in good condition for use in experiments.

[0045] Step 4: Cell grouping and treatment: BV2 cells in good growth condition were randomly divided into a blank (NC) control group, a model group (LPS group), and a piracetam (PIR)-containing serum group, a 5% Da Xie Fei Tang (low-dose Da Xie Fei Tang group, DXFT-L) group, a 10% Da Xie Fei Tang (medium-dose Da Xie Fei Tang group, DXFT-M) group, and a 20% Da Xie Fei Tang (high-dose Da Xie Fei Tang group, DXFT-H)-containing serum group. The BV2 cells in the serum-containing groups were pretreated for 4 hours. Except for the NC group, all other groups received 0.1 μg / mL of serum. -1 BV2 cells were stimulated with LPS for 24 hours. After different treatments, cell supernatant and cells were collected from each group as samples for subsequent experiments.

[0046] Step 5, CCK-8 cell viability assay: BV2 cells were administered at a rate of 1 × 10⁸ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates. After pretreatment with drug-containing serum for 4 hours, all groups except the NC group received 0.1 μg / mL of the drug-containing serum. -1 After culturing in LPS solution for 24 hours, 100 μL of 10% CCK8 reagent prepared with basal medium was added, and the mixture was incubated in an incubator for 30 minutes. The absorbance at 450 nm was measured using a microplate reader, and the cell viability (%) was calculated according to the formula: (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance) × 100%.

[0047] Step 6, Enzymatic determination of NO content: Collect cells from each treatment group under section 4, and determine the NO content according to cell number (10... 4 Add the extraction solution at a ratio of 1 mL / min, sonicate the cells on ice (200 W power, 3 s sonication, 7 s interval, total time 5 min), then centrifuge at 12000 r / min for 15 min at 4 °C, discard the precipitate, and place the supernatant on ice for testing. Perform the test according to the kit instructions.

[0048] Step 7: Detection of changes in inflammatory factor content by enzyme-linked immunosorbent assay (ELISA): Collect cell supernatant from each treatment group under item 4, and determine the content of IL-1β, IL-6, TNF-α, IL-4, IL-10, arachidonic acid (AA), prostaglandin E2 (PGE2), thromboxane A2 (TXA2), and leukotriene D4 (LTD4) according to the kit instructions;

[0049] Step 8: Detection of pro-inflammatory and anti-inflammatory factor mRNA expression levels by qRT-PCR: After BV2 cells were treated with LPS and serum, the supernatant was discarded, and total RNA was extracted from the cells using the Trizol method. After quantification, cDNA was synthesized by reverse transcription according to the kit instructions. cDNA was then used as a template for PCR amplification. PCR reaction conditions: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 25 s, 55℃ annealing for 35 s, 72℃ extension for 45 s, 35 cycles; 72℃ extension for 10 min. GAPDH was used as an internal control. -ΔΔCt The method was used to quantify relative mRNA expression levels; primer sequences are shown in Table 1.

[0050] Table 1 Primer sequences

[0051] Tab1 Primer sequences

[0052]

[0053]

[0054] Step 9: Western blotting to detect the expression levels of TLR4, HIF-1α, VEGF, iNOS, ALOX5, COX-2, Bcl-2, and Bax proteins: After modeling and stimulation with drug-containing serum, cells were collected, added with cell lysis buffer, and lysed on ice for 30 min at 4℃ and 12000 rpm. -1 Centrifuge for 30 min, collect cells, and determine the protein concentration of each group according to the experimental procedure of the BCA kit. Equal amounts of protein from each group are loaded for SDS-PAGE electrophoresis separation. After electrophoresis, transfer the proteins on the gel to a PVDF membrane, then block with 5% skim milk powder at room temperature for 2 h, wash three times with TBST, and then incubate the primary antibody at 4℃ overnight at 4:00 with the following dilution ratios: HIF-1a, iNOS, TLR4, VEGF, Bax, Bcl-2, and COX-2 diluted 1:1000, β-actin diluted 1:5000, and ALOX5 diluted 1:500. Wash the membrane three times with TBST; incubate with HRP-labeled secondary antibody (1:5000) at room temperature for 1 h; wash the membrane three times with TBST, add ECL chemiluminescence buffer to evenly cover the membrane surface, expose using a chemiluminescence imaging system, analyze the band gray values ​​using ImageJ software, and calculate the relative expression level of the target protein using β-actin as an internal reference.

[0055] Step 10: An Agilent 5975C-7890A GC-MS system was used; an Agilent HP-5MS quartz capillary column (0.25mm×30m, 0.25μm) was used, with an injection volume of 1μL and high-purity helium; the ion source temperature was 230℃, and the injection port temperature was 280℃; the initial column temperature was 70℃, held for 4 min, then 8℃ / min~125℃, 6℃ / min~200℃, and 4℃ / min~300℃, held for 5 min; the diaphragm purge flow rate was 3mL / min; the mass spectrometry ionization voltage was 70eV, and the full scan was performed at m / z 35-800.

[0056] Step 11, Statistical Methods: Experimental data were used... The results indicate that SPSS 15.0 statistical software was used for one-way ANOVA. Pairwise comparisons between multiple samples in the groups were first tested for homogeneity of variance. If the variances were homogeneous, the LSD method was used; otherwise, Dunnett's T3 method was used. The bar charts were drawn using GraphPad Prism 5 software.

[0057] Example 1: After BV2 cells were plated and allowed to adhere, each drug-treated group was pretreated with serum containing the drug for 4 hours, and then 0.1 μg / mL of serum was added. -1 The effects of LPS stimulation on BV2 cell viability after 24 hours are shown in the figure. Figure 1A. Compared with the NC group, the cell viability and NO content of BV2 cells in the LPS group were significantly decreased (P<0.01); compared with the LPS group, the cell viability of BV2 cells in each drug-containing serum group was significantly increased (P<0.05 or P<0.01), and the NO content was significantly decreased (P<0.01) (see...). Figure 1 B).

[0058] Example 2: ELISA and qRT-PCR detection results are as follows Figure 2 and 3 As shown, compared with the NC group, the LPS group significantly increased the secretion and mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in BV2 cells (P<0.01), and significantly decreased the secretion and mRNA expression levels of anti-inflammatory factors IL-4 and IL-10 (P<0.01). Compared with the LPS group, except for the DXFT-L group, all other serum-containing groups significantly decreased the LPS-induced secretion and mRNA expression levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in BV2 cells (P<0.05 or P<0.01), and significantly increased the secretion and mRNA expression levels of anti-inflammatory factors L-4 and IL-10 (P<0.05 or P<0.01).

[0059] Example 3, WB experimental results are as follows Figure 4 and 5 As shown, compared with the NC group, the expression of TLR4, HIF-1α, VEGF, iNOS, COX-2, ALOX5, and Bax proteins in BV2 cells of the LPS group was significantly increased (P<0.01), while the expression of Bcl-2 protein was significantly decreased (P<0.01). Compared with the LPS group, except for the DXFT-L group, the expression of TLR4, HIF-1α, VEGF, iNOS, COX-2, ALOX5, and Bax proteins in all other serum-treated groups was significantly decreased (P<0.01), while the expression of Bcl-2 protein was significantly increased (P<0.01). In addition, qRT-PCR results showed that the expression levels of COX-2 and iNOS mRNA also showed the same trend (P<0.01). Figure 5 ).

[0060] Example 4: ELISA experimental results are as follows Figure 6 As shown, compared with the NC group, the LPS group significantly increased the secretion of LTD4, PGE2 and TAX2 in BV2 cells (P<0.01) and decreased the secretion of AA (P<0.01); compared with the LPS group, each drug-containing serum group significantly decreased the secretion of LTD4, PGE2 and TAX2 induced by LPS in BV2 cells (P<0.05 or P<0.01) and significantly increased the secretion of AA (P<0.05 or P<0.01).

[0061] This invention utilizes LPS to induce BV2 cells to construct a neuroinflammation model. The experimental groups were: a blank control group, a model group, and a piracetam serum group (0.324 g / kg). -1 Cell viability was assessed using CCK-8 assay in serum containing 5%, 10%, and 20% Da Xie Fei Tang (a traditional Chinese medicine formula). Nitric oxide (NO) production in the cell supernatant was measured using a nitric oxide (NO) assay kit. The levels of interleukin (IL)-1, interleukin (IL)-6, tumor necrosis factor-α (TNF-α), interleukin (IL)-4, interleukin (IL)-10, arachidonic acid (AA), LTD4, PEG2, and TAX2 in the cell supernatant were detected by ELISA. Inducible nitric oxide synthase (NIOS) was detected by qRT-PCR. The mRNA expression levels of synthase-iNOS, cyclooxygenase-2 (COX-2), and pro-inflammatory and anti-inflammatory cytokines IL-6, IL-1β, TNF-α, IL-4, and IL-10 were measured using Western blot analysis. The results showed that both 10% and 20% serum containing Daxiefei Decoction significantly increased the activity of neuroinflammatory cells; significantly decreased the gene and protein expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in neuroinflammatory cells, and increased the gene and protein expression levels of anti-inflammatory cytokines IL-4 and IL-10; significantly downregulated the protein expression levels of TLR4, HIF-1α, VEGF, iNOS, COX-2, ALOX5, and Bax in neuroinflammatory cells, and significantly upregulated Bcl-2. The expression level of l-2 protein was significantly increased; the content of AA in inflammatory cells was significantly increased; and the content of its metabolites LTD4, PGE2, and TAX2 was significantly decreased. This study indicates that Da Xie Fei Tang has anti-neuroinflammatory and neuroprotective effects, and its mechanism of action can be achieved by regulating the HIF-1 / VEGF / AA signaling axis.

[0062] Example 5: The gas chromatography-mass spectrometry analysis results are shown in the figure. A total of 53 components were identified using the NIST17 database, including 17 amino acids, 8 organic acids, 6 sugars, 2 saturated fatty acids, 2 unsaturated fatty acids, 3 lipids, and 15 other components.

[0063] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A serum containing the medicinal formula Da Xie Fei Tang, which has anti-neuritis effects, characterized in that... The drug-containing serum inhibits the inflammatory response of microglia by regulating the HIF-1 / VEGF / AA signaling pathway, and its preparation method includes: (1) Take 20-94 parts of stir-fried Lepidium apetalum, 20-94 parts of rhubarb, 20-94 parts of white peony root, 7-32 parts of roasted licorice root, 7-32 parts of Scutellaria baicalensis and 7-32 parts of dried ginger, soak them in 8 times the amount of water for 1 hour, and then decoct them twice. (2) Combine the filtrates and concentrate them to a crude drug weight of 0.5-3 g / mL, then freeze-dry to obtain Da Xie Fei Tang freeze-dried powder; (3) Dissolve the freeze-dried powder of Da Xie Fei Tang in 10-40 g / kg -1 SPF-grade Wistar rats were administered the dose by gavage for 5 minutes to 7 days. (4) Blood was collected from the heart 5 min to 8 h after the last administration of the drug, and the serum was collected and freeze-dried to obtain the serum containing Da Xie Fei Tang.

2. The serum containing the medicated Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects according to claim 1, characterized in that, The concentration of the drug-containing serum used is 5%-20% (v / v).

3. The serum containing the medicated Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects according to claim 1, characterized in that, The anti-neuritis effect is manifested by LPS-induced decrease in BV2 cell viability and excessive secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α. Its anti-neuritis effect is confirmed by the following indicators: (1) The viability of BV2 cells increased by 29% to 62%; (2) Significantly reduced the mRNA expression levels and contents of pro-inflammatory factors IL-1β, IL-6, and TNF-α (P<0.01); (3) Significantly increased the mRNA expression level and content of anti-inflammatory factors IL-4 and IL-10 (P<0.01); (4) The content of pro-inflammatory mediator NO decreased by 7% to 15%.

4. The serum containing the medicated Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects according to claim 1, characterized in that, The modulation of the HIF-1 / VEGF / AA signal axis is as follows: The expression of TLR4, HIF-1α, VEGF, iNOS, ALOX5 and COX2 proteins was significantly downregulated (P<0.01); The levels of LTD4, PGE2, and TXA2 in the ALOX5 and COX2-mediated arachidonic acid (AA) metabolic pathway decreased by 12%–27%. The AA content increased by 4% to 22%.

5. The serum containing the medicated Da Xie Fei Tang (a traditional Chinese medicine formula) with anti-neuritis effects according to claim 1, characterized in that, The drug-containing serum simultaneously regulates apoptosis-related proteins: (1) Bax protein expression decreased by 18% to 78%; (2) Bc1-2 protein expression increased by 52% to 352%.

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