Application of combination of ligustrazine and hydroxysafflor yellow A in preparation of medicine for treating CIS ischemia reperfusion injury

Abstract

The invention provides application of combination of ligustrazine and hydroxysafflor yellow A in preparation of a medicine for treating CIS ischemia reperfusion injury, and belongs to the technical field of medicines. According to the application disclosed by the invention, ligustrazine and hydroxysafflor yellow A are found to have a synergistic effect, and the combination of ligustrazine and hydroxysafflor yellow A can inhibit a TLR4 / NF-kappa B mediated inflammation signal channel, activate an Nrf2 / HO-1 mediated oxidative stress signal channel, reduce damaged M1 type MG and increase protective M2 type MG, so that the application has the effects of inhibiting TLR4 / NF-kappa B mediated inflammation signal channel and inhibiting TLR4 / NF-kappa B mediated oxidative stress signal channel. The anti-inflammatory and anti-oxidative stress reactions of the BV2 cells induced by OGD / R are exerted, the activation amounts of different types of MG are regulated and controlled, and a scientific and reasonable basis is provided for solving the two important disease mechanisms of anti-inflammatory and anti-oxidative stress in ischemic cerebrovascular diseases and regulating and controlling the activation amounts of different types of MG.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of ligustrazine combined with hydroxysafflor yellow A in preparing a medicine for treating CIS ischemia-reperfusion injury. Background Art

[0002] Cerebral stroke, also known as stroke in traditional Chinese medicine, is a disease that seriously affects human health and has high morbidity, high disability, high mortality and high recurrence rates. Among them, cerebral ischemic stroke (CIS) accounts for 70% to 80% of stroke. After CIS occurs, multiple pathophysiological processes are involved, such as energy metabolism disorder, excitotoxicity, abnormal mitochondrial reaction, oxidative stress, inflammatory response, autophagy and abnormal immune response. These factors and mechanisms are interrelated and interact with each other, leading to cell apoptosis or neuronal necrosis in the ischemic area. Among them, the interaction between inflammation and oxidative stress response is the main cause of CIS ischemia-reperfusion injury (CIRI).

[0003] Microglial cells (MG) are resident immune cells of the central nervous system (CNS). After cerebral ischemia, MG are rapidly activated and proliferate, initiating an inflammatory response and releasing a variety of proinflammatory cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO), which aggravate inflammatory damage to the CNS. However, MG does not continuously secrete inflammatory factors and reactive oxygen species. Depending on the nature, intensity and duration of the stimulus, MG will show different activation states and phenotypes, namely the damaging M1 type and the protective M2 type, playing both harmful and beneficial roles, playing the role of a "double-edged sword". Therefore, after cerebral ischemia, how to inhibit inflammation and oxidative stress response and regulate the activation state of MG is an important way to reduce the damage after cerebral ischemia.

[0004] In recent years, thrombolytic therapy has been used in the clinical treatment of CIS, and its potential risks are also accompanied. For example, thrombolytic therapy can easily cause cerebral hemorrhage, reperfusion injury, and vascular reocclusion in patients. Therefore, it is particularly important to continue to explore drugs for the treatment of ischemic reperfusion injury in CIS. Traditional Chinese medicine has a long history of treating ischemic stroke. Chuanxiong and safflower are commonly used drugs for the treatment of ischemic stroke. Both are blood-activating and stasis-removing drugs. Chuanxiong mainly promotes blood circulation and qi, while safflower mainly promotes blood circulation and dissipates stasis. Studies have found that Chuanxiong's effective ingredient, ligustrazine, has the effect of resisting free radical damage, can reduce secondary damage caused by oxygen free radicals after brain injury, increase the activity of superoxide dismutase SOD in brain tissue, inhibit the increase of MDA in the brain after cerebral ischemia-reperfusion, and promote the recovery of brain nerve function. The safflower active ingredient, hydroxysafflor yellow A, can significantly downregulate the expression of TLR4 in middle cerebral artery occlusion / reperfusion (MCAO) model mice, inhibit the phosphorylation of NF-κB p-p65, ERE1 / 2, JNK and p38, and inhibit the secretion of inflammatory cytokines such as TNF-α and IL-1β. Although there are many studies on the effects of ligustrazine and hydroxysafflor yellow A on ischemic cerebrovascular disease, the focus is on the two important pathogenic mechanisms of anti-inflammatory and anti-oxidative stress in ischemic cerebrovascular disease and the activation of different types of MG. There are no reports on the combination of ligustrazine and hydroxysafflor yellow A in the treatment of CIS ischemia-reperfusion injury. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the use of ligustrazine combined with hydroxysafflor yellow A in the preparation of a drug for treating CIS ischemia-reperfusion injury. Ligustrazine and hydroxysafflor yellow A have a synergistic effect, which can effectively inhibit the inflammation and oxidative stress response in OGD / R-induced MG and regulate the activation amount of different types of MG, thereby treating CIS ischemia-reperfusion injury.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of ligustrazine combined with hydroxysafflor yellow A in preparing a medicine for treating CIS ischemia-reperfusion injury.

[0008] Preferably, the molar ratio of ligustrazine to hydroxysafflor yellow A is 1:(1-4).

[0009] Preferably, the ligustrazine is combined with hydroxysafflor yellow A to inhibit cell damage after glucose-oxygen deprivation / re-glucose-reoxygenation.

[0010] Preferably, the ligustrazine is combined with hydroxysafflor yellow A to reduce the level of inflammatory factors, and the inflammatory factors include IL-1β, TNF-α and IL-6.

[0011] Preferably, the ligustrazine is combined with hydroxysafflor yellow A to reduce NO and MDA levels.

[0012] Preferably, the ligustrazine is combined with hydroxysafflor yellow A to improve the SOD level.

[0013] Preferably, the ligustrazine combined with hydroxysafflor yellow A reduces the protein expression of TLR4 and NF-κB, and inhibits the TLR4 / NF-κB signaling pathway.

[0014] Preferably, the ligustrazine combined with hydroxysafflor yellow A increases the protein expression of Nrf2 and HO-1, and activates the Nrf2 / HO-1 signaling pathway.

[0015] Preferably, the ligustrazine combined with hydroxysafflor yellow A reduces the expression of iNOS, thereby reducing the damaging M1 type MG; and increases the expression of Arg-1, thereby increasing the protective M2 type MG.

[0016] The present invention also provides a medicine for treating CIS ischemia-reperfusion injury, wherein the medicine comprises ligustrazine and hydroxysafflor yellow A as active ingredients, and the molar ratio of ligustrazine to hydroxysafflor yellow A is 1:(1-4).

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The present invention provides the use of ligustrazine combined with hydroxysafflor yellow A in the preparation of a drug for treating CIS ischemia-reperfusion injury. The combined use of ligustrazine and hydroxysafflor yellow A can inhibit the TLR4 / NF-κB-mediated inflammatory signaling pathway, activate the Nrf2 / HO-1-mediated oxidative stress signaling pathway, reduce the damaging M1-type MG and increase the protective M2-type MG, exert anti-inflammatory and anti-oxidative stress reactions on BV2 cells induced by OGD / R, and regulate the activation amount of different types of MG, and the effect is significantly better than that of using them alone. The two have a synergistic effect, which provides a scientific and reasonable basis for solving the two important pathogenic mechanisms of anti-inflammatory and anti-oxidative stress in ischemic cerebrovascular disease and regulating the ischemia-reperfusion injury caused by the activation amount of different types of MG. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : The toxicity results of ligustrazine and hydroxysafflor yellow A on BV2 cells;

[0020] Figure 2 : The optimal drug concentration results of ligustrazine and hydroxysafflor yellow A after OGD / R injury;

[0021] Figure 3: The optimal drug concentration results of ligustrazine combined with hydroxysafflor yellow A after OGD / R injury;

[0022] Figure 4 :Effects of different drug administration groups on the inflammatory response of BV2 cells after OGD1.5 h / R12 h;

[0023] Figure 5 :Effects of different drug administration groups on the oxidative response of BV2 cells after OGD1.5 h / R12 h;

[0024] Figure 6 : Different doses of ligustrazine combined with hydroxysafflor yellow A affect the inflammatory response of BV2 cells after OGD1.5 h / R12 h;

[0025] Figure 7 : The results of different doses of ligustrazine combined with hydroxysafflor yellow A on the oxidative response of BV2 cells after OGD1.5 h / R12 h;

[0026] Figure 8 :WB results of TLR4 / NF-κB in different drug administration groups;

[0027] Fig. 9 :Effects of different drug administration groups on TLR4 / NF-κB signaling pathway in BV2 cells after OGD / R;

[0028] Fig.10 :WB results of Nrf2 / HO-1 in different drug administration groups;

[0029] Fig.11 :Effects of different drug administration groups on Nrf2 / HO-1 signaling pathway in BV2 cells after OGD / R;

[0030] Fig.12 :The results of cell slide immunofluorescence;

[0031] Fig.13 : Effects of ligustrazine combined with hydroxysafflor yellow A on the expression of iNOS and Arg-1. DETAILED DESCRIPTION

[0032] The present invention provides the use of ligustrazine combined with hydroxysafflor yellow A in the preparation of a drug for treating CIS ischemia-reperfusion injury. The molar ratio of ligustrazine to hydroxysafflor yellow A is 1:(1-4), preferably 1:1 or 1:2 or 1:4. The ligustrazine combined with hydroxysafflor yellow A of the present invention inhibits cell damage after glucose-oxygen deprivation / re-glucose-reoxygenation, and the dosage of ligustrazine in protecting microglia induced by glucose-oxygen deprivation / re-glucose-reoxygenation and reducing inflammation and oxidative stress is preferably 1-30 μmol·L -1, Hydroxysafflor yellow A 0.2~25μmol·L -1 , more preferably 2.5 μmol·L -1 Hydroxysafflor yellow A+5μmol·L -1 Ligustrazine, 2.5 μmol·L -1 Hydroxysafflor yellow A+10μmol·L -1 Ligustrazine, 5 μmol·L -1 Hydroxysafflor yellow A+5μmol·L -1 Ligustrazine or 5 μmol·L -1 Hydroxysafflor yellow A+10μmol·L -1 Ligustrazine.

[0033] The present invention uses an OGD / R BV2 cell model to obtain that ligustrazine combined with hydroxysafflor yellow A has good anti-inflammatory and antioxidant effects, and the two have a synergistic effect. The present invention finds that the combination of two monomers of ligustrazine and hydroxysafflor yellow A has a protective effect on OGD / R damaged BV2 cells, and can significantly reduce the expression of inflammatory factors such as IL-1β, IL-6, TNF-α, and the levels of oxidative stress indicators such as NO and MDA. After ligustrazine combined with hydroxysafflor yellow A acted on OGD / RBV2 cells, the level of M1 type MG marker iNOS decreased, the expression of cell NF-κB and TLR4 decreased, the expression of M2 type MG marker Arg-1 increased, and the expression of cell Nrf2 and HO-1 increased. Ligustrazine combined with hydroxysafflor yellow A can inhibit the TLR4 / NF-κB-mediated inflammatory signaling pathway, activate the Nrf2 / HO-1-mediated oxidative stress signaling pathway, reduce the damaging M1 type MG and increase the protective M2 type MG. This shows that Ligustrazine combined with hydroxysafflor yellow A can reduce inflammation, oxidative stress response and regulate the activation amount of different types of MG, and has the effect of treating CIS ischemia-reperfusion injury.

[0034] The present invention also provides a drug for treating CIS ischemia-reperfusion injury, wherein the drug has ligustrazine and hydroxysafflor yellow A as active ingredients, and the molar ratio of ligustrazine and hydroxysafflor yellow A is 1: (1-4), preferably 1: 1 or 1: 2 or 1: 4. Preferably, the drug of the present invention also includes a pharmaceutically acceptable carrier and / or excipient to ensure the convenient preparation and clinical application of the prepared pharmaceutical preparation. Further preferably, the pharmaceutical excipients of the present invention include but are not limited to at least one of a diluent, a wetting agent, an adhesive, a lubricant, a colorant, and a coating agent. The dosage form of the drug of the present invention includes tablets, capsules, granules, pills, injections, suspensions, dispersants, etc. The dosage form of the drug of the present invention is not limited thereto, and other dosage forms that can be achieved are within the scope of protection of the present invention.

[0035] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] In the specific embodiment of the present invention, BV2 cells were purchased from Wuhan Pronocell Life Science Co., Ltd.; hydroxysafflor yellow A (batch number: ST02370120) and ligustrazine (batch number: ST20370120) were purchased from Shanghai Standard Standard Technology Service Co., Ltd.; Cell Counting Kit-8 detection kit (MA0218-1) was purchased from Dalian Meilun Biotechnology Co., Ltd.; mouse TNF-α (ml002095), mouse IL-1β (ml098416), and mouse IL-6 (ml098430) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.; total superoxide dismutase (T-SOD) test kit (A001-1-1), nitric oxide (NO) assay kit (A012-1-2), and trace malondialdehyde (MDA) assay kit (A003-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute; NF-κB antibody (AF5006) and TLR4 antibody (AF7017) were purchased from Affinity Company; Nrf2 antibody (80593-1) was purchased from Wuhan Sanying Biotechnology Co., Ltd.; HO-1 antibody (sc-390991) was purchased from Santa Cruz Biotechnology; Tubulin antibody (M03989-3) was purchased from Boster Biological Company; goat anti-rabbit secondary antibody (BST16K26C16K55) and goat anti-mouse secondary antibody (BST15K18B15K51) were purchased from Thermo Company, USA; inducible nitric oxide synthase (iNOS) was purchased from Abcam; arginase-1 (Arg-1) antibody was purchased from Cell Signaling Technology.

[0037] GraphPad Prism 9.0 was used for data analysis. The quantitative data were expressed as x±s. The t-test was used for pairwise comparison. One-way analysis of variance (ANOVA) was used for comparison of multiple groups of data. P<0.05 was considered statistically significant.

[0038] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] 1. Screening of safe concentration range of monomer drugs

[0042] (1) In vitro culture of BV2 cells and construction of inflammation and oxidative stress models:

[0043] After adding an appropriate amount of complete culture medium (containing 1% penicillin-streptomycin antibody and 10% fetal bovine serum) to the culture dish, 1×10 5 cell·mL -1 The BV2 cell resuspension was evenly inoculated into a 10 cm cell culture dish and placed in a 37°C, 5% CO2 cell culture incubator for culture. When the cell growth density reached 80%, the cell medium was replaced. 5 ml of basal culture medium was added to the culture dish of the normal group, and 100 ng ml of basal culture medium was added to the model group. -1 Lipopolysaccharide (LPS) and 20ng ml -1 5 ml of TNF-α recombinant protein working solution was added; the cells were cultured at 37°C and 5% CO2 for 24 h and 12 h respectively to establish LPS and TNF-α-induced inflammation and oxidative stress models.

[0044] (2) CCK-8 kit screens the safe concentration range of each monomer drug:

[0045] Hydroxysafflor yellow A and ligustrazine were dissolved in DMSO solution and prepared into 100 μmol·L -1 , 500μmol·L -1 , 2.5mmol·L -1 、12.5mmol·L -1 , 50mmol·L -1 Before adding the drug, the mother solution was diluted with complete culture medium to a final concentration of 0.2 μmol·L -1 , 1μmol·L -1 , 5μmol·L -1 , 25μmol·L -1 , 100 μmol·L -1 The working solution of concentration gradient should ensure that the DMSO content in the working solution is ≤2‰. 5 cell·mL -1The BV2 cell resuspension was inoculated into a 96-well culture plate (200 μl per well). After 6 hours of adherence culture, the culture medium was discarded, and 200 μl of drug-containing working solution was added to each well (2‰ DMSO was added to the model control group). Three replicates were made for each monomer drug concentration, and normal cells were set as the control group. The cells were cultured for 24 hours, and the cell medium was replaced with DMEM complete medium. 10 μl of cck8 solution was added to each well, and the culture was continued at 37°C and 5% CO2 for 1 hour. The culture plate was taken out and the absorbance of the cells was measured by an automatic microplate reader at a wavelength of 450nm. The cell survival rate was calculated based on the absorbance values ​​of the normal group and the model group cells, and the non-toxic concentration range of the drug was determined. If there was no statistical difference in cell proliferation at this concentration, it was a non-toxic concentration.

[0046] Cell viability = (OD 样本 -OD 正常 ) / (OD 模型对照 -OD 正常 )×100%.

[0047] The results are as follows Figure 1 As shown in the results, the concentration of hydroxysafflor yellow A (HSYA) was ≤5 μmol·L -1 When the concentration of ligustrazine (TMP) was ≤25μmol·L -1 It was not toxic to BV2 cells.

[0048] 2. OGD / R model establishment and efficacy verification

[0049] (1) Cell grouping and establishment of OGD / R model:

[0050] BV2 cells in the logarithmic growth phase were randomly divided into a normal group (cultured as usual), an OGD / R modeling group (including an OGD / R group, an OGD / R+monomer 1, an OGD / R+monomer 2, an OGD / R+effective component combined use group, and an OGD / R model group). The normal group was cultured in a conventional manner. The OGD / R modeling method was as follows: after culturing at 37°C, 5% CO2 for 24 hours, the culture medium was discarded, rinsed with PBS, and an appropriate amount of sugar-free DMEM was added and placed in an anaerobic box. After 1.5 hours, the culture dish was removed, the waste liquid was aspirated and discarded, and an appropriate amount of complete culture medium was added. The modeling was continued at 37°C, 5% CO2 for 12 hours (reperfusion culture). Before OGD, the corresponding drugs were added to the OGD / R group, OGD / R+monomer 1, OGD / R+monomer 2, and OGD / R+effective component combined use group. The cell growth was observed under an inverted microscope, and the cell culture supernatant and cells were collected for later use.

[0051] (2) Effects of ligustrazine, hydroxysafflor yellow A and their combination on BV2 cells damaged by OGD / R

[0052] Before OGD, the OGD / R+monomer 1 group was added with a final concentration of 0 μmol·L -1 , 1μmol·L -1 , 2.5 μmol·L -1 , 5μmol·L -1 , 10μmol·L -1 , 15μmol·L -1 , 20μmol·L -1 , 25μmol·L -1 , 30μmol·L -1 The final concentration of ligustrazine in the OGD / R+monomer group was 0 μmol·L -1 , 0.2μmol·L -1 , 1μmol·L -1 , 2.5 μmol·L -1 , 5μmol·L -1 , 10μmol·L -1 , 15μmol·L -1 , 20μmol·L -1 , 25μmol·L -1 Hydroxysafflor yellow A; OGD / R + active ingredient combination group were added to the sugar-free medium at a final concentration of 2.5 μmol·L -1 Hydroxysafflor yellow A+5μmol·L -1 Ligustrazine, 2.5 μmol·L -1 Hydroxysafflor yellow A+10μmol·L -1 Ligustrazine, 5 μmol·L -1 Hydroxysafflor yellow A+5μmol·L -1 Ligustrazine, 5 μmol·L -1 Hydroxysafflor yellow A+10μmol·L -1 The OGD / R modeling method was the same as step (1), and the cell viability was determined using the CCK-8 kit.

[0053] The results of the optimal drug concentrations of ligustrazine and hydroxysafflor yellow A after OGD / R injury are as follows Figure 2 As shown in Figure 2, the optimal drug concentration of ligustrazine combined with hydroxysafflor yellow A after OGD / R injury is as follows Figure 3 As shown in the figure, TMP represents ligustrazine, HSYA represents hydroxysafflor yellow A; compared with the model group, ***P<0.001, **P<0.01, *P<0.05 (x±s, n=3). The results showed that the drug concentration of ligustrazine was 5μmol·L -1 , 10μmol·L -1The drug concentration of hydroxysafflor yellow A was 2.5 μmol·L -1 , 5μmol·L -1 When the BV2 cells were treated with a two-by-two combination, the four doses of ligustrazine and hydroxysafflor yellow A did not show cytotoxic effects on BV2 cells damaged by OGD / R. The results suggest that the combination of ligustrazine and hydroxysafflor yellow A has a protective effect on BV2 cells damaged by OGD / R.

[0054] (3) ELISA detection of related cytokines

[0055] Inflammatory cytokine detection:

[0056] Before OGD, the OGD / R+HSYA group was supplemented with a final concentration of 5 μmol·L -1 Hydroxysafflor yellow A, OGD / R+TMP group were added to the sugar-free medium at a final concentration of 10 μmol·L -1 In the OGD / R+HSYA+TMP group, the final concentration of 5 μmol·L -1 hydroxysafflor yellow A and 10 μmol·L -1 The OGD / R modeling method was the same as step (1), the OGD / R group was the model group, and the normal group was the normal group.

[0057] The collected cell supernatant was taken and operated according to the instructions of the ELISA detection kit to detect the inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) in the culture medium, and the absorbance value of each monitoring index was measured at 450nm on an enzyme marker, and the concentration of each factor in the sample was calculated according to the standard curve.

[0058] Results of the inflammatory response of BV2 cells in different drug groups after OGD1.5 h / R12 h Figure 4 As shown in the figure, ***P<0.001, **P<0.01, *P<0.05 (x±s, n=3). The IL-1β concentration in the OGD / R+HSYA group was 89.47±0.99 pg·mL -1 , the IL-1β concentration in the OGD / R+TMP group was 80.04±0.37pg·mL -1 , the IL-1β concentration in the OGD / R+HSYA+TMP group was 67±1.49 pg·mL -1 ; The TNF-α concentration in the OGD / R+HSYA group was 479.15±1.17pg·mL -1, the TNF-α concentration in the OGD / R+TMP group was 428.24±3.90pg·mL -1 , the TNF-α concentration in the OGD / R+HSYA+TMP group was 393.88±2.75pg·mL -1 ; The IL-6 concentration in the OGD / R+HSYA group was 126.54±1.82pg·mL -1 , the IL-6 concentration in the OGD / R+TMP group was 113.45±0.7pg·mL -1 , the IL-6 concentration in the OGD / R+HSYA+TMP group was 91.4±1.03pg·mL -1 . The results showed that compared with the normal group, the levels of inflammatory factors such as IL-1β, TNF-α, and IL-6 in the model group were increased (P<0.001); compared with the model group, the levels of inflammatory factors in the OGD / R+hydroxysafflor yellow A group, OGD / R+ligustrazine group, and OGD / R+hydroxysafflor yellow A+ligustrazine group were all downregulated, and the differences were statistically significant. The level of inflammatory factors in the OGD / R+hydroxysafflor yellow A+ligustrazine group was lower than that in the OGD / R+hydroxysafflor yellow A group and the OGD / R+ligustrazine group, and the differences were significant. It suggests that the combined use of ligustrazine and hydroxysafflor yellow A has a good anti-inflammatory effect on OGD / R-damaged BV2 cells and the effect is better than the use of the two drugs alone.

[0059] Detection of oxidative stress related indicators:

[0060] Before OGD, the OGD / R+HSYA group was supplemented with a final concentration of 5 μmol·L -1 Hydroxysafflor yellow A, OGD / R+TMP group were added to the sugar-free medium at a final concentration of 5 μmol·L -1 In the OGD / R+HSYA+TMP group, the final concentration of 5 μmol·L -1 hydroxysafflor yellow A and 5 μmol·L -1 The OGD / R modeling method was the same as step (1), the OGD / R group was the model group, and the normal group was the normal group.

[0061] The collected cell supernatant was taken and operated according to the instructions of the ELISA detection kit to detect the content of oxidative stress-related indicators superoxide dismutase (T-SOD), nitric oxide (NO), and trace malondialdehyde (MDA) in the culture medium, and the absorbance value of each monitoring indicator was measured at 450nm on an enzyme marker, and the concentration of each factor in the sample was calculated according to the standard curve.

[0062] The results of the oxidative response of BV2 cells after OGD1.5 h / R12 h in different drug groups are as follows Figure 5 As shown in the figure, ***P<0.001, **P<0.01, *P<0.05 (x±s, n=3). The NO concentration in the OGD / R+HSYA group was 76.92±2.11 μmol·L -1 The NO concentration in the OGD / R+TMP group was 82.29±2.1μmol·L -1 The NO concentration in the OGD / R+HSYA+TMP group was 61.6±1.58μmol·L -1 ; The SOD concentration in the OGD / R+HSYA group was 1.54±0.01U·mL -1 , the SOD concentration in the OGD / R+TMP group was 1.47±0.01U·mL -1 The SOD concentration in the OGD / R+HSYA+TMP group was 1.86±0.03U·mL -1 ; The MDA concentration in the OGD / R+HSYA group was 7.39±0.06nmol·mL -1 , the MDA concentration in the OGD / R+TMP group was 10.30±0.08nmol·mL -1 , the MDA concentration in the OGD / R+HSYA+TMP group was 6.19±0.15nmol·mL -1 The results showed that the levels of oxidative stress factors such as NO and MDA in the model group were significantly higher than those in the normal group, while the level of SOD was significantly lower than that in the normal group, and the differences were statistically significant; compared with the model group, the levels of oxidative stress-related indicators such as NO and MDA in the OGD / R+hydroxysafflor yellow A group, OGD / R+ligustrazine group, and OGD / R+hydroxysafflor yellow A+ligustrazine group were significantly reduced, and the level of SOD was increased, and the differences were statistically significant; the levels of NO and MDA in the OGD / R+hydroxysafflor yellow A+ligustrazine group were significantly lower than those in the OGD / R+hydroxysafflor yellow A group and OGD / R+ligustrazine group, and the level of SOD was higher than those in the OGD / R+hydroxysafflor yellow A group and OGD / R+ligustrazine group. This suggests that the combined use of ligustrazine and hydroxysafflor yellow A has a better antioxidant effect on BV2 cells after OGD / R than the use of either drug alone.

[0063] Effect test of different dose groups of ligustrazine combined with hydroxysafflor yellow A:

[0064] Before OGD, each group added 2.5 μmol·L -1 Hydroxysafflor yellow A+5μmol·L -1 Ligustrazine (denoted as OGD / R+2.5+5), 2.5 μmol·L -1 Hydroxysafflor yellow A+10μmol·L -1Ligustrazine (denoted as OGD / R+2.5+10), 5 μmol·L -1 Hydroxysafflor yellow A+5μmol·L -1 Ligustrazine (represented as OGD / R+5+5), 5 μmol·L -1 Hydroxysafflor yellow A+10μmol·L -1 Ligustrazine (denoted as OGD / R+5+10); the OGD / R modeling method was the same as step (1), the OGD / R group was the model group, and the normal group was the normal group.

[0065] The results of different doses of ligustrazine combined with hydroxysafflor yellow A on the inflammatory response of BV2 cells after OGD1.5 h / R12 h are shown in Figure 6 The results of different doses of ligustrazine combined with hydroxysafflor yellow A on the oxidative response of BV2 cells after OGD1.5h / R12h are shown in Figure 7 As shown in the figure, compared with the model group, ***P<0.001, **P<0.01, *P<0.05 (x±s, n=3). The results showed that compared with the model group, 5μmol·L -1 +10μmol·L -1 The levels of IL-1β and TNF-α in the 5 μmol·L -1 +5μmol·L -1 The NO level of the group was significantly downregulated, and the levels of SOD oxidative stress-related indicators were significantly upregulated, both showing statistical significance (P<0.001).

[0066] (4) Western blot detection

[0067] TLR4 / NF-κB signaling pathway related detection:

[0068] Before OGD, the OGD / R+HSYA group was supplemented with a final concentration of 5 μmol·L -1 Hydroxysafflor yellow A, OGD / R+TMP group were added to the sugar-free medium at a final concentration of 10 μmol·L -1 In the OGD / R+HSYA+TMP group, the final concentration of 5 μmol·L -1 hydroxysafflor yellow A and 10 μmol·L -1 The OGD / R modeling method was the same as step (1), the OGD / R group was the model group, and the normal group was the normal group.

[0069] Collect cells, add lysis buffer to lyse for 30 minutes, centrifuge at 4℃ (12000rpm, 15min), extract total cell protein, and detect protein concentration by BCA method. According to the molecular weight of the target protein, 7.5%, 10%, and 12.5% ​​gel SDSPAGE electrophoresis were used. After the protein was transferred to the PVDF membrane by wet transfer, it was blocked with 5% skim milk powder and blocked on a shaker at room temperature for 1h. Nuclear factor κB (NF-κB) antibody (1:1000) and Toll-like receptor 4 (TLR4) antibody (1:1000) were added respectively; incubated overnight at 4℃. The next day, the diluted corresponding secondary antibody was added and incubated at room temperature for 1h. After washing the membrane, the scanner was used for development, and the protein band pictures were imported into Image J software for processing to obtain the gray value of each protein band. Finally, the data were imported into Prism software for statistical analysis.

[0070] The WB results of TLR4 / NF-κB in different drug groups are as follows Figure 8 The effects of different drug administration groups on the TLR4 / NF-κB signaling pathway in BV2 cells after OGD / R are shown in Fig. 9 As shown in the figure, ***P<0.001, **P<0.01, *P<0.05 (x±s, n=3). The results showed that compared with the normal group, the expression of TLR4 and NF-κB in the model group was significantly increased (P<0.001); compared with the model group, the expression of TLR4 and NF-κB in the OGD / R+hydroxysafflor yellow A+ligustrazine group was significantly decreased (P<0.001), and the expression was lower than that in the OGD / R+hydroxysafflor yellow A group and the OGD / R+ligustrazine group. The results showed that the combination of hydroxysafflor yellow A and ligustrazine can inhibit the inflammatory response of BV2 cells induced by OGD / R through the TLR4 / NF-κB signaling pathway, and the inhibitory effect is better than the single use of hydroxysafflor yellow A or ligustrazine.

[0071] Related detection of Nrf2 / HO-1 signaling pathway:

[0072] Before OGD, the OGD / R+HSYA group was supplemented with a final concentration of 5 μmol·L -1 Hydroxysafflor yellow A, OGD / R+TMP group were added to the sugar-free medium at a final concentration of 5 μmol·L -1 In the OGD / R+HSYA+TMP group, the final concentration of 5 μmol·L -1 hydroxysafflor yellow A and 5 μmol·L -1 The OGD / R modeling method was the same as step (1), the OGD / R group was the model group, and the normal group was the normal group.

[0073] Collect cells, add lysis buffer to lyse for 30 minutes, centrifuge at 4°C (12000rpm, 15min), extract total cell protein, and detect protein concentration by BCA method. According to the molecular weight of the target protein, 7.5%, 10%, and 12.5% ​​gel SDSPAGE electrophoresis were used. After the protein was transferred to the PVDF membrane by wet transfer, it was blocked with 5% skim milk powder and blocked on a shaker at room temperature for 1h. Nuclear transcription factor NF-E2 related factor 2 (Nrf2) antibody (1:2000), heme oxygenase-1 (HO-1) antibody (1:500), Tubulin antibody (1:8000) were added respectively, and incubated at 4°C overnight. The next day, the diluted corresponding secondary antibody was added and incubated at room temperature for 1h. After washing the membrane, the scanner was used for development, and the protein band pictures were imported into Image J software for processing to obtain the gray value of each protein band. Finally, the data were imported into Prism software for statistical analysis.

[0074] The WB results of Nrf2 / HO-1 in different drug groups are as follows Fig.10 The effects of different drug administration groups on the Nrf2 / HO-1 signaling pathway in BV2 cells after OGD / R are shown in Fig.11 As shown in the figure, ***P<0.001, **P<0.01, *P<0.05 (x±s, n=3). The results showed that the expression of Nrf2 and HO-1 in the model group was significantly increased compared with the normal group (P<0.001); compared with the model group, the expression of Nrf2 and HO-1 in the OGD / R+hydroxysafflor yellow A+ligustrazine group was significantly increased (P<0.001); the Nrf2 and HO-1 in the OGD / R+hydroxysafflor yellow A+ligustrazine group were significantly higher than those in the OGD / R+hydroxysafflor yellow A group and the OGD / R+ligustrazine group (P<0.01). The results suggest that the combination of hydroxysafflor yellow A and ligustrazine inhibits OGD / R-induced oxidative stress in BV2 cells by activating the Nrf2 / HO-1 signaling pathway, and the activation effect is better than that of hydroxysafflor yellow A or ligustrazine alone.

[0075] (5) Immunofluorescence detection

[0076] Before OGD, the OGD / R+HSYA group was supplemented with a final concentration of 5 μmol·L -1 Hydroxysafflor yellow A, OGD / R+TMP group were added to the sugar-free medium at a final concentration of 10 μmol·L -1 In the OGD / R+HSYA+TMP group, the final concentration of 5 μmol·L -1 hydroxysafflor yellow A and 10 μmol·L -1 The OGD / R modeling method was the same as step (1), the OGD / R group was the model group, and the normal group was the normal group.

[0077] BV2 cells were cultured on slides for 24 h, fixed with paraformaldehyde for 30 min, washed with PBS, and then inducible nitric oxide synthase (iNOS) antibody (1:300) and arginase-1 (Arg-1) antibody (1:300) were added, incubated at 4°C overnight, incubated at 37°C in the dark, secondary antibody was added, and DAPI solution was added to cover the slides in the dark. Images were captured under a fluorescence microscope and imported into Image J software for processing and analysis to obtain the fluorescence intensity values ​​of each group. Finally, the data were imported into Prism software for statistical analysis.

[0078] The results of immunofluorescence on cell slides are as follows Fig.12 The results of the combined use of ligustrazine and hydroxysafflor yellow A on the expression of iNOS and Arg-1 are shown in Fig.13 As shown. The results showed that compared with the normal group, the expression of M1 marker iNOS in the model group cells was significantly increased (P<0.001); compared with the model group, the expression of iNOS in the OGD / R+hydroxysafflor yellow A+ligustrazine group cells was significantly reduced (P<0.001); the expression of iNOS in the OGD / R+hydroxysafflor yellow A+ligustrazine group cells was significantly lower than that in the OGD / R+hydroxysafflor yellow A group and the OGD / R+ligustrazine group, and the differences were statistically significant (P<0.01). It shows that the combination of hydroxysafflor yellow A and ligustrazine can downregulate the expression of the damaging M1 marker iNOS and inhibit the inflammatory and oxidative reactions of BV2 cells induced by OGD / R, and the effect is better than that of hydroxysafflor yellow A or ligustrazine alone. Compared with the normal group, the expression of Arg-1, a marker of M2, in the model group was significantly increased (P<0.001); compared with the model group, the expression of Arg-1 in the OGD / R+hydroxysafflor yellow A+ligustrazine group was significantly increased (P<0.001); the expression of Arg-1 in the OGD / R+hydroxysafflor yellow A+ligustrazine group was significantly increased compared with the OGD / R+hydroxysafflor yellow A group and the OGD / R+ligustrazine group (P<0.001 or P<0.01). Immunofluorescence results confirmed that the combination of hydroxysafflor yellow A and ligustrazine could upregulate the expression of Arg-1, a protective marker of M2, promote the transformation of MG from M1 to M2, and inhibit the inflammatory and oxidative reactions of BV2 cells induced by OGD / R, and the effect was better than that of hydroxysafflor yellow A or ligustrazine alone.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of ligustrazine combined with hydroxysafflor yellow A in the preparation of drugs for the treatment of CIS ischemia-reperfusion injury.

2. The use according to claim 1, characterized in that: The molar ratio of ligustrazine to hydroxysafflor yellow A is 1:(1-4).

3. The use according to claim 1, characterized in that: The ligustrazine combined with hydroxysafflor yellow A inhibits cell damage after glucose-oxygen deprivation / re-glucose-reoxygenation.

4. The use according to claim 1, characterized in that: The ligustrazine combined with hydroxysafflor yellow A reduces the level of inflammatory factors, including IL-1β, TNF-α and IL-6.

5. The use according to claim 1, characterized in that: The combination of ligustrazine and hydroxysafflor yellow A reduces the levels of NO and MDA.

6. The use according to claim 1, characterized in that: The ligustrazine combined with hydroxysafflor yellow A improves the SOD level.

7. The use according to claim 1, characterized in that: The ligustrazine combined with hydroxysafflor yellow A reduces the protein expression of TLR4 and NF-κB, and inhibits the TLR4 / NF-κB signaling pathway.

8. The use according to claim 1, characterized in that: The ligustrazine combined with hydroxysafflor yellow A increases the protein expression of Nrf2 and HO-1, and activates the Nrf2 / HO-1 signaling pathway.

9. The use according to claim 1, characterized in that: The combination of ligustrazine and hydroxysafflor yellow A reduces the expression of iNOS and reduces the damaging M1 type MG; increases the expression of Arg-1 and increases the protective M2 type MG.

10. A drug for treating CIS ischemia-reperfusion injury, characterized in that: The drug has ligustrazine and hydroxysafflor yellow A as active ingredients, and the molar ratio of ligustrazine to hydroxysafflor yellow A is 1:(1-4).

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