Pharmaceutical composition for improving myocardial injury by adjusting autoimmunity and application

Abstract

The invention discloses a pharmaceutical composition for improving myocardial injury by regulating autoimmunity and application, and relates to the technical field of biological medicines. The pharmaceutical composition for improving myocardial injury by adjusting autoimmunity comprises paeonol and pachymaran, the mass ratio of the paeonol to the pachymaran is (1-2): (1-2), and the invention further provides application of the pharmaceutical composition for improving myocardial injury by adjusting autoimmunity in preparation of drugs for treating myocardial injury. The traditional Chinese medicine composition can improve myocardial fibrosis, regulate CD4 + T cell immune balance and promote angiogenesis, and effectively solves the problems of poor myocardial repair effect after reperfusion injury after myocardial infarction and immune imbalance in a chronic inflammation stage after myocardial infarction.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a drug combination and its application that improves myocardial injury by regulating autoimmunity. Background Technology

[0002] Effectively reducing the incidence of reperfusion injury after myocardial infarction and promoting myocardial repair is a pressing clinical challenge. Inflammatory-immune responses are involved in the myocardial injury repair process following ischemia-reperfusion injury. This includes early, severe, and transient acute inflammation, as well as subsequent chronic inflammation. Because the inflammatory response in myocardial ischemia-reperfusion injury both exacerbates the damage and is necessary to clear necrotic tissue and promote repair, the treatment strategy is not to block inflammation, but rather to regulate it. The chronic inflammatory phase after myocardial infarction plays a crucial role in myocardial repair and is currently a key focus of research in this field.

[0003] Previous studies have shown that during the subsequent myocardial repair process, which is primarily characterized by chronic inflammation, the damaged area is dominated by acquired immunity. This is because CD4+ is involved during this period. + T cells are characterized by their diversity in differentiation and function, capable of differentiating into various subsets and producing different immune effects. They help regulate the immune response and maintain immune homeostasis. The imbalance between naïve CD4+ T cells, which differentiate into helper T cells (Th1 / Th2) and Th17, and regulatory T cells (Tregs), is closely related to the development and progression of myocardial repair after myocardial infarction. During myocardial ischemia-reperfusion injury, CD4+ T cells play a crucial role. + T cells have a dual role: when differentiated into Th1 cells, they can secrete interferon-γ to exacerbate inflammatory damage, while Treg cells can suppress inflammation by secreting substances similar to interleukin-10. Therefore, research is needed on how to regulate CD4. + T cell differentiation and maintaining the balance of cell subsets such as Th1 / Th2 and Th17 / Treg are of great significance for post-myocardial infarction treatment.

[0004] Traditional Chinese medicine (TCM), as a treasure of traditional culture, is crucial to national health and the inheritance of civilization. Researchers screened commonly used TCM formulas and drugs for the clinical treatment of ischemic heart disease and found that the use rate and efficacy of the peony bark-poria cocos combination were quite remarkable. It appeared in famous formulas such as Liuwei Dihuang Wan, Guizhi Fuling Wan, and Xiaoyao Wan, with a 1:1 ratio of the two. Poria cocos enters the Qi (vital energy) while peony bark enters the blood, regulating both Qi and blood, treating both the heart and lungs simultaneously, and achieving the effect of promoting blood circulation and removing blood stasis. Paeonol is the main active ingredient of peony bark, and poria cocos polysaccharide is the main active ingredient of poria cocos. Paeonol has significant antioxidant, anti-inflammatory, cardiomyocyte protective, and blood flow improving effects. Poria cocos polysaccharide has significant immunomodulatory functions and exerts anti-tumor and cardiovascular protective functions through immune regulation. Both peeonol and poria cocos polysaccharide play important roles in adaptive immune regulation; peeonol and poria cocos polysaccharide can affect CD4... + T cell proliferation and differentiation can regulate the balance between inflammation and immunity and improve myocardial ischemia, but the effect of combining these two drugs, and whether they can enhance efficacy and reduce toxicity, is currently unknown. This invention aims to find an effective drug combination that can reduce myocardial damage and promote myocardial repair. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a pharmaceutical composition and its application that improves myocardial injury by regulating autoimmunity, effectively solving the problems of high incidence of reperfusion injury after myocardial infarction, poor myocardial repair, and immune imbalance during the chronic inflammatory phase after myocardial infarction.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is: to provide a pharmaceutical composition that improves myocardial injury by regulating autoimmunity, comprising paeonol and poria cocos polysaccharide.

[0007] Furthermore, the mass ratio of paeonol to poria cocos polysaccharide is 1-2:1-2.

[0008] Furthermore, the mass ratio of paeonol to poria cocos polysaccharide is 1:1.

[0009] Furthermore, myocardial injury refers to myocardial injury following myocardial infarction.

[0010] A pharmaceutical formulation for treating myocardial injury, comprising the above-described pharmaceutical composition for improving myocardial injury by modulating autoimmunity and pharmaceutically acceptable excipients.

[0011] Furthermore, the pharmaceutical preparation is an oral preparation.

[0012] Application of pharmaceutical compositions that improve myocardial injury by modulating autoimmunity in the preparation of drugs for treating myocardial injury.

[0013] Furthermore, the aforementioned drugs for treating myocardial injury are CD4 inhibitors.+ Drugs that promote excessive activation of T cells.

[0014] Furthermore, the aforementioned drugs for treating myocardial injury are those that reduce the proportion of pro-inflammatory Th1 cells and / or increase the proportion of anti-inflammatory Treg cells.

[0015] Furthermore, the aforementioned drugs for treating myocardial injury are drugs that promote angiogenesis.

[0016] Furthermore, the aforementioned drugs for treating myocardial injury are drugs that improve the degree of myocardial fibrosis.

[0017] In summary, the present invention has the following beneficial effects: 1. This invention, based on the disease progression pattern after myocardial infarction, focuses on the interaction between immunity and inflammation during the transition from acute to chronic inflammation. The potential time points for drug action are set at two stages: the (sub)acute phase (3-10 days) and the chronic phase (0-28 days). Intervention with paeonol and poria cocos polysaccharide demonstrates that the synergistic effect of paeonol and poria cocos polysaccharide can reduce myocardial fibrosis after myocardial infarction, with better results in the chronic phase than the acute phase. Furthermore, the combined effect of the two drugs is superior to that of single drugs. Regarding angiogenesis, the combined use is superior to single administration in the chronic phase, indicating that combined therapy is more suitable for the chronic inflammatory phase after myocardial infarction. (The text then mentions CD4 in acquired immunity.) + T cell differentiation and the Th1 / Treg balance play a crucial role in myocardial injury and repair. Drug intervention significantly inhibited the excessive activation of CD4+ T cells, especially in the chronic phase, and also had a significant effect on CD4+ T cells. + The drug significantly inhibits the pro-inflammatory Th1 cells differentiated from T cells, and in both the acute and chronic phases, combination therapy is superior to monotherapy. For CD4... + Treg cells, which have anti-inflammatory effects after T cell differentiation, show a certain degree of enhancement after drug intervention, with significant differences in the acute phase, and combination therapy is superior to single-drug therapy.

[0018] 2. This invention combines paeonol and poria cocos polysaccharide, and their synergistic effect in the chronic phase after myocardial infarction is more significant than that of either drug alone. Furthermore, they can improve myocardial damage by regulating autoimmunity, which has promising application prospects for future drug development. Attached Figure Description

[0019] Figure 1 Figure 1 shows the effects of paeonol and poria cocos polysaccharide, used alone and in combination, on collagen fibers after myocardial reperfusion injury in mice. Figure 2 Flow cytometry analysis of CD4+ T cells 28 days after drug administration; Figure 3 The effect of the drug on CD4 28 days after administration + The effect of T cells is shown in the figure; Figure 4 CD4 in the early stage of reperfusion (3-10 days) + T-cell flow cytometry analysis diagram; Figure 5 For early reperfusion (3-10 days) drug effects on CD4 + The effect of T cells is shown in the figure; Figure 6 Flow cytometry analysis of Th1 cells 28 days after drug administration; Figure 7 The graph shows the effect of the drug on Th1 cells 28 days after administration. Figure 8 Flow cytometry analysis of Th1 cells in the early stage of reperfusion (3-10 days); Figure 9 Figure showing the effect of drugs on Th1 cells in the early stage of reperfusion (3-10 days); Figure 10 Flow cytometry analysis of Treg cells 28 days after drug administration; Figure 11 The graph shows the effect of the drug on Treg cells 28 days after administration. Figure 12 Flow cytometry analysis of Treg cells in the early stage of reperfusion (3-10 days); Figure 13 Figure showing the effect of drugs on Treg cells in the early stage of reperfusion (3-10 days); Figure 14 The graph shows the effect of the drug on α-SMA 28 days after administration; Figure 15 Figure showing the effect of drugs on α-SMA in the early stage of reperfusion (3-10 days); Figure 16 The graph shows the effect of the drug on TGF-β1 28 days after administration; Figure 17 The figure shows the effect of drugs on TGF-β1 in the early stage of reperfusion (3-10 days). Detailed Implementation

[0020] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0021] Example 1: Establishment of a mouse model of myocardial ischemia-reperfusion injury Mice were fasted for 8 hours before surgery. After weighing, anesthesia was induced using isoflurane, and a small animal ventilator was simultaneously turned on. The parameters were adjusted: peak airway pressure was set to 25 cm H2O, respiratory rate to 110 breaths / min, and ventilation rate to 0.8 L / min. After anesthesia induction, the anesthetic was connected to the ventilator, and the mice were intubated and connected to the ventilator to maintain respiration. The consistency between the mice's respiratory fluctuations and the ventilator frequency was observed. The mice were placed supine on a circulating heated operating table, their limbs fixed, and an electrocardiogram (ECG) was connected to record ECG changes during surgery. After preparing and disinfecting the surgical area, the skin near the 3rd and 4th intercostal spaces on the left side of the sternum was cut, and the subcutaneous tissue and muscles were bluntly dissected layer by layer to open the thoracic cavity. Once the heart was fully exposed, the pericardium was gently torn open. After exposing the left atrial appendage, a 6-0 suture needle was used to puncture the superficial myocardium 1 mm below the lower edge of the atrial appendage. A 0.2-0.3 cm 10-gauge polyethylene tube was inserted below the suture and ligated. Whitening of the left ventricular anterior wall myocardium, restricted local contractile movement, and distended left atrial appendage were observed. The electrocardiogram showed persistent ST-segment elevation with a convex shape, indicating successful myocardial ischemia. After successful modeling, ischemia was allowed to continue for 45 minutes, the polyethylene tube was removed, the chest was closed, and reperfusion was restored. Once the mice regained consciousness, the endotracheal tube was removed, and they were placed in a 38°C incubator overnight.

[0022] Example 2: Treatment with paeonol, poria cocos polysaccharide, and paeonol + poria cocos polysaccharide After one week of acclimatization, mice were randomly divided into four groups: blank control (NC) group, sham operation group, ischemia-reperfusion (I / R) group, ischemia-reperfusion + paeonol (I / R+PAE) group, ischemia-reperfusion + poria cocos polysaccharide (I / R+PCP) group, ischemia-reperfusion + paeonol, and poria cocos polysaccharide combined group (I / R+PAE+PCP). Specifically, after surgery, the paeonol group and poria cocos polysaccharide group were given different concentrations of paeonol aqueous solution (100 mg / kg, 50 mg / kg, 25 mg / kg) and poria cocos polysaccharide aqueous solution (100 mg / kg, 50 mg / kg, 25 mg / kg) by gavage. The combined drug group was given different concentrations of paeonol + poria cocos polysaccharide (100+100 mg / kg, 50+50 mg / kg, 25+25 mg / kg) by gavage according to the 1:1 ratio of the two drugs in the classic formula. Samples were collected at two different time points: one was taken on day 28 after surgery via gavage; the other was taken on day 10 after surgery via gavage starting on day 3.

[0023] Example 3 Masson staining After anesthesia and euthanasia, the heart was harvested and fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin, and serially sectioned to a thickness of approximately 5 μm. After dewaxing and hydration, Weigert's ferrugin staining solution was applied to cover the sections for staining for 5 min, followed by washing with ultrapure water to remove excess staining solution. Differentiation solution was applied for 5 s, followed by washing with ultrapure water for 30 s. Masson's blue solution was applied for blue reversion for 3 min, followed by washing with ultrapure water for 30 s. Ponceau S and fuchsin staining solutions were applied for 5 min, followed by washing with weak acid working solution for 30 s. Excess liquid was discarded, and phosphomolybdic acid was applied for 1 min, followed by washing with weak acid working solution for 30 s. Excess liquid was discarded, and aniline blue staining was applied for 30 s, followed by washing with weak acid working solution for 30 s. The sections were dehydrated, mounted, and the degree of myocardial fibrosis was observed under an optical microscope. The effects of paeonol and poria cocos polysaccharide, alone and in combination, on collagen fibers after myocardial reperfusion injury in mice were investigated. Figure 1 As shown. Figure 1 In the table, A represents the effect of the drug on myocardial collagen fibers 28 days after administration; B represents the effect of the drug on myocardial collagen fibers in the early stage of reperfusion (3-10 days). (n=3, vs Model ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0.0001) Depend on Figure 1 It was found that after 4 weeks of continuous administration, the degree of myocardial fibrosis was significantly improved in the treatment group, with the 100 mg / kg combination therapy group showing a more significant improvement. A drug concentration of 100 mg / kg was selected as the optimal concentration, and administration was started 3 days post-surgery and continued until day 10. Both the paeonol group and the combination therapy group significantly reduced collagen fiber content, and the combination therapy group was significantly superior to the Poria cocos polysaccharide monotherapy group.

[0024] Example 4: Effects of paeonol, poria cocos polysaccharide alone and in combination on CD4 levels in mice after myocardial reperfusion injury. + The effect of T cells After digestion and grinding of the myocardium, myocardial immune cells were isolated and prepared into a single-cell suspension. After mixing by pipetting and aspiration, the suspension was transferred to a 96-well U-shaped plate. FACS buffer was prepared by adding FBS (1:200) to PBS. Then, 150-200 μL of FACS buffer was added to each well, and the plate was centrifuged at 2000 rpm for 2 min at 4 °C, discarding the supernatant. Simultaneously, fluorescent antibodies were diluted with FACS buffer to prepare a cell surface antibody mixture. After washing, the antibody mixture was added to the 96-well U-shaped plate, and the antibody and cells were thoroughly mixed. The plate was then incubated at 4 °C in the dark for 15-20 min. After surface staining, cell membrane permeabilization was performed. After cell permeabilization and fixation, 200 μL of FACS buffer and 1×Wash Buffer were added sequentially, and the plate was centrifuged at 2200 rpm for 2 min at 4 °C, discarding the supernatant. Then, 30 μL / well of intracellular antibody mixture was added to the plate. After mixing by pipetting and aspiration, the plate was incubated at 4 °C in the dark for 1 h. After staining, add 200 μL of FACS buffer to each well, centrifuge, discard the supernatant, wash away unbound antibodies, resuspend the cells in 200 μL of FACS buffer per well, and transfer the cells to flow cytometry tubes through a 200-mesh filter. Perform flow cytometry analysis. The effects of paeonol, poria cocos polysaccharide alone and in combination on CD4+ in mice after myocardial reperfusion injury. + The effects of T cells, such as Figures 2-4 As shown. Among them, Figure 2 CD4 28 days after administration + Flow cytometry analysis of T cells Figure 2 In the middle, the horizontal axis is 10. 0 10 2 10 4 10 6 The vertical axis is -10. 3 ,0,10 3 10 4 10 5 . Figure 3 The effect of the drug on CD4 28 days after administration + The influence of T cells. Figure 4 CD4 in the early stage of reperfusion (3-10 days) + T-cell flow cytometry analysis diagram Figure 4 In the diagram, the horizontal axis represents 0, 50K, 100K, and 150K respectively; the vertical axis represents -10 respectively. 4 ,0,10 4 10 5 . Figure 5 The effect of drugs on CD4+ T cells in the early stage of reperfusion (3-10 days). (n=6, vs Model ns, P>0.05; ****, P<0.0001) Depend on Figures 2-5 It can be seen that, compared with the normal group, the proportion and number of CD4+ T cells in the myocardium of mice in the model group were significantly increased at both time points; compared with the model group, the proportion and number of CD4+ T cells in the myocardium of mice in each drug administration group were significantly decreased.

[0025] Example 5: Effects of Paeonol, Poria cocos polysaccharide alone and in combination on Th1 and Treg levels in mice after myocardial reperfusion injury. CD4 + After activation, T cells differentiate into pro-inflammatory Th1 cells and anti-inflammatory Treg cells.

[0026] (1) Effects of paeonol and poria cocos polysaccharide, alone and in combination, on Th1 cells after myocardial reperfusion injury in mice, such as Figures 6-9 As shown, where, Figure 6 This is a flow cytometry analysis of Th1 cells 28 days after drug administration. Figure 6 In the diagram, the horizontal coordinates are 0 and 10 respectively. 4 10 5 The vertical axis is 0, 10 respectively. 3 10 4 10 5 . Figure 7 The effect of the drug on Th1 cells 28 days after administration. Figure 8 This is a flow cytometry analysis of Th1 cells during the early stages of reperfusion (3-10 days). Figure 8 In the middle, the horizontal axis is 10. -3 10 0 10 3 10 6 10 9 10 12 The vertical axis is -10. 3 ,0,10 3 10 4 10 5 . Figure 9 The effect of drugs on Th1 cells in the early stage of reperfusion (3-10 days). (n=6, vs Model ns, P>0.05; *, P<0.05; ***, P<0.001; ****, P<0.0001) Depend on Figures 6-9 It can be seen that, compared with the normal group, the proportion and number of Th1 cells in the myocardium of mice in the model group were significantly increased at both time points; compared with the model group, the proportion and number of Th1 cells in the myocardium of mice in each drug administration group were significantly decreased, and the combined drug administration group was significantly better than the single drug group.

[0027] (2) Effects of paeonol and poria cocos polysaccharide, alone and in combination, on Treg cells after myocardial reperfusion injury in mice, such as Figures 10-13 As shown, where, Figure 10 This is a flow cytometry analysis of Treg cells 28 days after drug administration. Figure 10 In the middle, the horizontal axis is 10. 0 10 2 10 4 10 6 The vertical axis is -10. 2 ,0,10 2 10 3 10 4 . Figure 11 The effect of the drug on Treg cells 28 days after administration. Figure 12 This is a flow cytometry analysis of Treg cells in the early stage of reperfusion (3-10 days), with the horizontal axis representing 10... 0 10 2 10 4 10 6 10 8 The vertical axis is 10. 0 10 1 10 2 10 3 10 4 10 5 . Figure 13 The effect of drugs on Treg cells in the early stage of reperfusion (3-10 days). (n=6, vs Model ns, P>0.05, *; ****, P<0.0001) Depend on Figures 10-13 It can be seen that, compared with the normal group, the proportion of Treg cells in the myocardium of mice in the model group decreased at both time points; compared with the model group, the proportion and number of Treg cells in the myocardium of mice in each drug administration group increased significantly, and the combined drug administration group was better than the single drug group.

[0028] Example 6: Effects of Paeonol, Poria cocos polysaccharide alone and in combination on angiogenesis after myocardial reperfusion injury in mice. α-SMA (α-smooth muscle actin) is a marker of vascular smooth muscle, while TGF-β1 (a marker of tissue repair and fibrosis) is an important cytokine that regulates cell growth, proliferation, differentiation, and apoptosis, and plays a leading role in tissue repair and fibrosis. In the early stages of myocardial infarction, it is moderately upregulated, inhibiting excessive inflammation and stabilizing cardiac structure. In the middle and late stages (repair and remodeling phase), it is persistently highly expressed, strongly driving myofibroblast activation and collagen deposition, leading to cardiac stiffness and heart failure.

[0029] Mouse hearts were fixed in 4% paraformaldehyde, dehydrated, embedded, sectioned, dewaxed, and hydrated. Antigen retrieval was performed using a heat retrieval method, followed by 5% blocking serum. Sections were blocked at room temperature for 1 h. For antibody preparation, 0.5 μg of primary antibody was mixed with 1 μL of FlexLinker, and FlexBuffer was added to bring the volume to 8 μL. The mixture was gently mixed and incubated at room temperature in the dark for 5 min. 2 μL of FlexQuencher was added, mixed, and incubated at room temperature in the dark for 5 min. PBS was added to bring the volume to 50-100 μL, and the mixture was incubated overnight at 4°C in the dark. After rinsing with PBS, DAPI-containing mounting media were incubated at room temperature for 60 min, and observed under a fluorescence microscope. The effects of paeonol, poria cocos polysaccharide alone and in combination on angiogenesis after myocardial reperfusion injury in mice were investigated. Figures 14-17 As shown, where, Figure 14 To determine the effect of the drug on α-SMA 28 days after administration, Figure 15 To investigate the effects of drugs on α-SMA during the early stages of reperfusion (3-10 days), Figure 16 To determine the effect of the drug on TGF-β1 28 days after administration, Figure 17 The effect of drugs on TGF-β1 in the early stage of reperfusion (3-10 days). (n=3, vs Model ns, P>0.05; *, P<0.05; ***, P<0.001; ****, P<0.0001) Depend on Figures 14-17 It was found that in the long-term intervention group, α-SMA expression increased after drug administration. The effects of Poria cocos polysaccharide alone and the combination of Paeonia lactiflora and Poria cocos polysaccharide were significantly different from the model group, and the combination therapy was superior to the single-drug therapy. However, in the early post-myocardial infarction period, single-drug therapy was superior to the combination therapy, indicating that the combination therapy is more suitable for the chronic phase after myocardial infarction. Immunofluorescence results of TGF-β1 showed that in the long-term intervention group, drug administration significantly reduced TGF-β1 expression, indicating effective inhibition of excessive myocardial fibrosis, with the combination therapy being superior to the single-drug group. However, no significant changes were observed in the early post-myocardial infarction period.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pharmaceutical composition for improving myocardial damage by modulating autoimmunity, characterized by, The paeonol and the pachymaran are in a mass ratio of 1-2:1-2.

2. The pharmaceutical composition for improving myocardial injury by modulating autoimmunity according to claim 1, wherein The myocardial injury is myocardial injury after myocardial infarction.

3. The pharmaceutical composition for improving myocardial damage by modulating autoimmunity according to claim 1, wherein the compound is a compound represented by the following formula 1: ###0001### Formula 1 The pharmaceutical composition for improving myocardial injury by regulating autoimmunity and the pharmaceutically acceptable adjuvant are as claimed in claim 1.

4. A pharmaceutical preparation for treating myocardial damage, characterized by, The pharmaceutical preparation is an oral preparation.

5. The pharmaceutical preparation for treating myocardial injury according to claim 4, wherein 6. Use of the pharmaceutical composition for improving myocardial injury by regulating autoimmunity as claimed in claim 1 in the preparation of a medicament for treating myocardial injury. The medicament for treating myocardial injury is a medicament for reducing the proportion of proinflammatory Th1 cells or / and increasing the proportion of anti-inflammatory Treg cells.

7. Use according to claim 6, wherein The drug for treating myocardial injury is a CD4 + a drug for inhibiting T cell overactivation.

8. The use according to claim 6, wherein the compound is ###0002### The medicament for treating myocardial injury is a medicament for promoting angiogenesis.

9. The use according to claim 6, wherein The medicament for treating myocardial injury is a medicament for improving the degree of myocardial fibrosis.

10. The use according to claim 6, wherein ​

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