Composition for reducing stem cell transplantation rejection and application

Abstract

The invention relates to the field of biological medicine, in particular to a composition for reducing stem cell transplantation rejection and application. The composition is combined with the peptide I, the peptide II and the compound extract group, so that the proliferation and activation capacities of T cells can be reduced. By combining with a specific receptor on the surface of a T cell, a calmodulin / NFAT pathway is blocked, and secretion of cell factors such as IFN-gamma and IL-2 is reduced, so that proliferation and activation of the T cell are inhibited. Meanwhile, the composition can induce generation of regulatory T cells and inhibitory T cells, promote differentiation of undifferentiated T cells to Treg cells by up-regulating expression of TGF-beta and IDO, and enhance the inhibition function of the Treg cells; th1 / Th2 cytokine secretion can also be regulated, so that Th1 type cytokine secretion is reduced, Th2 type cytokine secretion is increased, and body immune tolerance is induced.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to compositions and applications for reducing stem cell transplant rejection. Background Technology

[0002] Stem cell transplantation has shown great potential in the treatment of various diseases, but transplant rejection severely impacts the success rate and patient prognosis. While traditional immunosuppressants have some effect, they also have serious side effects, such as cytotoxicity, weakened immunity leading to infection, diabetes, and kidney dysfunction. Therefore, developing safe and effective methods and compositions to reduce stem cell transplant rejection is of great significance. Summary of the Invention

[0003] Natural extracts have become a research hotspot due to their wide availability and relatively few side effects; however, there are currently no reports of innovative combinations of specific peptides I and II with compound extracts in this field. The purpose of this invention is to provide a composition that reduces stem cell transplant rejection. Through multi-target synergistic regulation of the immune response, it effectively reduces transplant rejection, improves the success rate of stem cell transplantation, and possesses both safety and novelty.

[0004] In a first aspect, the present invention provides a composition for reducing stem cell transplant rejection, comprising peptide I: Val-Glu-Ser-Thr-Pro-Arg-Ala-Gly, peptide II: Leu-Asn-Gly-Lys-Met-Glu-Ser-His, and a compound extract. The compound extract is composed of *Sinomenium acutum*, *Paeonia lactiflora*, and *Angelica sinensis* in a mass ratio of 3:2:1, wherein the average weight content of sinomenine is ≥15%, the average weight content of paeoniflorin is ≥10%, and the average weight content of ferulic acid is ≥8%.

[0005] Peptide I combined with peptide II and the compound extract can reduce the proliferation and activation of T cells. By binding to specific receptors on the surface of T cells, it blocks the calcineurin / NFAT pathway, reduces the secretion of cytokines such as IFN-γ and IL-2, thereby inhibiting T cell proliferation and activation. Simultaneously, this composition can induce the production of regulatory T cells (Tregs) and suppressor T cells, promote the differentiation of undifferentiated T cells into Treg cells by upregulating the expression of TGF-β and IDO, and enhance the suppressive function of Treg cells; it can also regulate the secretion of Th1 / Th2 cytokines, reducing the secretion of Th1 cytokines (such as IFN-γ) and increasing the secretion of Th2 cytokines (such as IL-4 and IL-10), inducing immune tolerance. Furthermore, peptide I can increase the number of NK cells and NKT cells in vivo, enhancing the body's immune surveillance and immunomodulatory capabilities. Peptide I is prepared using chemical methods such as solid-phase synthesis or recombinant expression methods.

[0006] Peptide II, combined with peptide I and the compound extract, mimics the spatial conformation of natural immunomodulatory peptides, binding to relevant receptors on the surface of dendritic cells and inhibiting dendritic cell maturation. Specifically, it downregulates the expression of CD80, CD86, and MHC class II molecules on the surface of dendritic cells, blocking their transmission of activation signals to T cells, thereby synergistically enhancing the induction of immune tolerance. Peptide II is prepared using chemical methods such as solid-phase synthesis or recombinant expression.

[0007] The compound extracts, including peptides I and II, can inhibit the proliferation of autosensitized T cells and reduce the release of pro-inflammatory cytokines (such as TNF-α and IL-6) by inhibiting the NF-κB signaling pathway. Paeoniflorin can inhibit delayed-type hypersensitivity immune responses, regulate T cell function, and promote the production of Treg cells. Ferulic acid can reduce TNF-α levels and has antioxidant and anti-inflammatory effects, alleviating inflammatory responses and tissue damage at the transplantation site. The preparation process of the compound extract group includes: mixing 300g of Sinomenium acutum, 200g of Paeonia lactiflora, and 100g of Angelica sinensis, adding 6000mL of 70% ethanol, and refluxing and extracting 3 times (2 hours each time). The extracts are combined and concentrated under reduced pressure until there is no alcohol odor. The solution is diluted with water to 1000mL and loaded onto a macroporous adsorption resin column (e.g., AB-8 type). The column is eluted with 1000mL of water first, and then with 2000mL of 50% ethanol. The ethanol eluent is collected, concentrated, and freeze-dried to obtain the compound extract. The average weight content of Sinomenine is ≥15%, the average weight content of paeoniflorin is ≥10%, and the average weight content of ferulic acid is ≥8%.

[0008] In some embodiments, the composition comprises, by weight, 10-20 parts of peptide I, 5-15 parts of peptide II, and 20-40 parts of a compound extract.

[0009] In some embodiments, the composition comprises, by weight, 15 parts of peptide I, 10 parts of peptide II, and 30 parts of a compound extract.

[0010] In some embodiments, the composition contains 1.5 mg / mL of peptide I, 1.0 mg / mL of peptide II, and 3.0 mg / mL of a compound extract.

[0011] The weight proportions mentioned in the above embodiments can be scaled up proportionally, and the solvent system can be adjusted according to the route of administration (such as 5% glucose solution for intravenous injection), all of which fall within the scope of protection of this invention.

[0012] Furthermore, the present invention also provides the use of the above composition in the preparation of formulations that reduce stem cell transplant rejection.

[0013] Furthermore, the present invention also provides the use of the above composition in the preparation of formulations that directly inhibit effector T cell activation, induce immune tolerance, and regulate immune homeostasis.

[0014] Furthermore, the present invention also provides the use of the above composition in the preparation of formulations that downregulate co-stimulatory molecules and reduce the secretion of pro-inflammatory factor IL-12.

[0015] Furthermore, the present invention also provides the use of the above composition in the preparation of a formulation that inhibits the expression of pro-inflammatory factors TNF-α and IL-6 and promotes the proliferation of Treg cells.

[0016] Beneficial effects: The composition of this invention, through the innovative combination of peptide I, peptide II, and a compound extract group, achieves multi-target synergistic regulation of the immune response. Compared with existing single-component or simple combination solutions, it has a more significant effect in reducing stem cell transplantation rejection. The overall composition is derived from natural sources or artificially synthesized under controlled conditions, with relatively few side effects, providing a new, effective, and safe approach to improving the success rate of stem cell transplantation. Attached Figure Description

[0017] Figure 1 The T cell proliferation inhibition rate of each group in Example 1 is given by: "***" P < 0.001, "**" P < 0.01, "*" P < 0.05.

[0018] Figure 2 The values ​​represent the IFN-γ expression inhibition rate and p-NFAT protein expression inhibition rate for each group in Example 2. "***" P < 0.001, "**" P < 0.01, "*" P < 0.05.

[0019] Figure 3 The percentages (%) of CD4⁺CD25⁺Foxp3⁺Treg expressing cells, NK expressing cells, and NKT expressing cells in each group of Example 2 are given. “***” P<0.001, “**” P<0.01, “*” P<0.05.

[0020] Figure 4 The percentage reduction (%) of CD80-expressing cells, CD86-expressing cells, and MHCII-expressing cells in each group of Example 3 is given by: “***” P<0.001, “**” P<0.01, “*” P<0.05.

[0021] Figure 5 The percentage decrease in relative IL-12 mRNA expression in each group of Example 3 is 10%. "***" P < 0.001, "**" P < 0.01.

[0022] Figure 6 The percentage of TNF-α expression reduction in each group of Example 4 is denoted as '***', where P < 0.001 and '**' are P < 0.01.

[0023] Figure 7 The percentage of IL-6 expression reduction in each group of Example 4 is given by “**” P < 0.01 and “*” P < 0.05.

[0024] Figure 8 For Example 4, the percentage increase of Treg cells in each group was detected by flow cytometry (%). "**" P<0.01, "*" P<0.05. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Reagents not specifically described in detail in this invention are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be obtained from the prior art.

[0026] Example 1: Composition Formulation Validation Test 1. Preparation and grouping of test samples Seven experimental groups were set up, with six parallel samples in each group, and the following processing was performed: Peptide I group: Prepare a 1.5 mg / mL peptide I solution using sterile physiological saline (0.9% NaCl). Peptide II group: A 1.0 mg / mL peptide II solution was prepared using sterile physiological saline (0.9% NaCl). Compound extract group: A solution containing 3.0 mg / mL of compound extract was prepared using a 50% ethanol-physiological saline mixture (V:V=1:1). Peptide I + Peptide II group: First, prepare peptide I and peptide II solutions separately, then mix them to obtain a mixed solution containing 1.5 mg / mL peptide I and 1.0 mg / mL peptide II. Peptide I + Compound Extract Group: First, prepare peptide I solution and compound extract solution separately, then mix them to obtain a mixed solution containing 1.5 mg / mL peptide I and 3.0 mg / mL compound extract. Peptide II + Compound Extract Group: First, prepare peptide II solution and compound extract solution separately, then mix them to obtain a mixed solution containing 1.0 mg / mL peptide II and 3.0 mg / mL compound extract. Composition group: First, prepare peptide I solution, peptide II solution and compound extract solution separately, and then mix them to obtain a composition solution containing 1.5 mg / mL peptide I, 1.0 mg / mL peptide II and 3.0 mg / mL compound extract group.

[0027] 2. Test methods Using 96-well cell culture plates, 100 μL of allogeneic T cell suspension (cell density 1 × 10⁶ cells / well) was added to each well. 6cells / mL, that is, 1×10⁻⁶ cells / mL per well 5 Add 10 μL of the corresponding sample solution (to ensure a total volume of 110 μL and avoid liquid overflow from the wells), and repeat the process 6 times per group. Incubate at 37°C and 5% CO2 for 48 hours.

[0028] 3. Experimental Data and Results like Figure 1 The figures show the inhibition rates of T cell proliferation by peptide I, peptide II, compound extract, peptide I + peptide II, peptide I + compound extract, peptide II + compound extract, and the combined composition group. As can be seen from the figure, the T cell proliferation inhibition rate of the combined composition group was significantly higher than that of the other groups.

[0029] The results of this experiment show that the composition provided by this invention has a significantly better inhibitory effect on T cell proliferation than single components or pairwise combinations. Its inhibition rate reached 70.3%, nearly 40% higher than the optimal pairwise combination (the combination of peptide I and the compound extract, with an inhibition rate of 50.5%). This demonstrates a synergistic effect between peptide I, peptide II, and the compound extract. This synergistic effect overcomes the limitations of single components or simple combinations, proving that the three-component combination can enhance the inhibitory ability on immune effector cells through a complementary mechanism, thus laying the foundation for reducing transplant rejection.

[0030] Example 2: Validation experiment on the mechanism of action of T cells 1. Test sample The seven groups of solutions are the same as in Example 1, namely: Peptide I group: 1.5 mg / mL peptide I solution; Peptide II group: 1.0 mg / mL peptide II solution; Compound extract group: 3.0 mg / mL compound extract solution; Peptide I + Peptide II group: 1.5 mg / mL peptide I + 1.0 mg / mL peptide II mixed solution; Peptide I + Compound extract group: 1.5 mg / mL peptide I + 3.0 mg / mL compound extract mixed solution; Peptide II + Compound extract group: 1.0 mg / mL peptide II + 3.0 mg / mL compound extract mixed solution; Composition group: 10 mg / mL composition solution of the present invention (including 1.5 mg / mL peptide I, 1.0 mg / mL peptide II, and 3.0 mg / mL compound extract).

[0031] 2. Test methods Add 1 mL of human peripheral blood T cells (2 × 10⁶ cells) to each well of a 24-well cell culture plate. 6 The samples were divided into a control group (with an equal volume of physiological saline) and a treatment group (with 100 μL of the corresponding sample solution added to each group (total volume 1.1 mL). Each group had 6 replicates and were incubated at 37°C for 24 hours.

[0032] The levels of IFN-γ mRNA were detected by RT-qPCR, and the IFN-γ expression inhibition rate was calculated. The IFN-γ expression inhibition rate was the rate of change in the IFN-γ mRNA level of each group of samples relative to the control group.

[0033] Western blot was used to detect calcineurin p-NFAT protein, with rabbit anti-human p-NFAT antibody as the primary antibody at a dilution of 1:1000. The p-NFAT protein expression inhibition rate was calculated, which was the rate of change in the amount of p-NFAT protein in each sample compared to the control group.

[0034] Flow cytometry was used to detect CD4⁺CD25⁺Foxp3⁺Treg (antibodies: APC-CD4, PE-CD25, FITC-Foxp3), NK cells (CD3⁻CD56⁺), and NKT cells (CD3⁺CD56⁺).

[0035] 3. Experimental Data and Results like Figure 2 The figures show the inhibition rates of IFN-γ expression in human peripheral blood T cells by peptide I, peptide II, compound extract, peptide I + peptide II, peptide I + compound extract, peptide II + compound extract, and the combined composition group. As can be seen from the figures, the inhibition rate of IFN-γ expression in the combined composition group was significantly higher than that in the other groups.

[0036] like Figure 2 The inhibition rates of p-NFAT protein expression in human peripheral blood T cells by peptide I group, peptide II group, compound extract group, peptide I + peptide II group, peptide I + compound extract group, peptide II + compound extract group, and combined group are also shown. As can be seen from the figure, the inhibition rate of p-NFAT protein expression in the combined group is significantly higher than that in the other groups.

[0037] This experiment confirms that the complete composition provided by this invention can regulate T cell function through multiple targets. The composition provided by this invention can reduce p-NFAT levels and block the calcineurin / NFAT pathway, thereby reducing the secretion of IFN-γ pro-inflammatory factors and directly inhibiting effector T cell activation.

[0038] like Figure 3 The figures show the proportions of CD4⁺CD25⁺Foxp3⁺Treg cells, NK cells, and NKT cells in the peptide I group, peptide II group, compound extract group, peptide I + peptide II group, peptide I + compound extract group, peptide II + compound extract group, and combined group after treatment with human peripheral blood T cells. As can be seen from the figures, the proportions of CD4⁺CD25⁺Foxp3⁺Treg cells, NK cells, and NKT cells in the combined group were significantly higher than those in the other groups.

[0039] This confirms that the composition provided by the present invention can also promote the proliferation of CD4⁺CD25⁺Foxp3⁺Treg cells, NK cells and NKT cells, causing their proliferation to reach or even exceed 2.0 times, inducing immune tolerance, and thereby enhancing immune regulation function.

[0040] The above mechanisms collectively demonstrate that the composition provided by this invention has the dual regulatory advantage of inhibiting effector cells and enhancing regulatory cells, which is more advantageous than the one-sided effect of a single component.

[0041] Example 3: Verification experiment on the mechanism of action against dendritic cells 1. Test sample The seven groups of solutions are the same as in Example 1, namely: Peptide I group: 1.5 mg / mL peptide I solution; Peptide II group: 1.0 mg / mL peptide II solution; Compound extract group: 3.0 mg / mL compound extract solution; Peptide I + Peptide II group: 1.5 mg / mL peptide I + 1.0 mg / mL peptide II mixed solution; Peptide I + Compound extract group: 1.5 mg / mL peptide I + 3.0 mg / mL compound extract mixed solution; Peptide II + Compound extract group: 1.0 mg / mL peptide II + 3.0 mg / mL compound extract mixed solution; Composition group: 10 mg / mL composition solution of the present invention (including 1.5 mg / mL peptide I, 1.0 mg / mL peptide II, and 3.0 mg / mL compound extract).

[0042] 2. Test methods Using 24-well cell culture plates, 1 mL of human monocyte-induced dendritic cells (1 × 10⁶) were added to each well. 6 Add 100 μL of the corresponding sample solution (total volume 1.1 mL) to each well, with 6 replicates per group, and incubate for 48 hours. A control group was also set up, with an equal volume of physiological saline added.

[0043] The expression ratios of CD80, CD86, and MHCII were detected by flow cytometry (antibodies: PE-CD80, APC-CD86, FITC-HLA-DR). The expression reduction rate of CD80, CD86, and MHCII was calculated. The expression reduction rate is the change rate of the expression ratios of CD80, CD86, and MHCII in each group of samples relative to the control group.

[0044] IL-12 mRNA levels were detected by RT-qPCR, and the IL-12 mRNA expression inhibition rate was calculated. The IL-12 mRNA expression inhibition rate was the rate of change in IL-12 mRNA levels in each group of samples relative to the control group.

[0045] 3. Experimental Data and Results like Figure 4The figures show the proportions of CD80, CD86, and MHCII-expressing cells in the peptide I group, peptide II group, compound extract group, peptide I + peptide II group, peptide I + compound extract group, peptide II + compound extract group, and combined group after treatment with human monocyte-induced dendritic cells. As shown in the figure, the proportions of CD80, CD86, and MHCII-expressing cells in the combined group were significantly higher than those in the other groups.

[0046] like Figure 5 The relative expression levels of IL-12 mRNA in human monocyte-induced dendritic cells are shown in the figures for peptide I, peptide II, compound extract, peptide I + peptide II, peptide I + compound extract, peptide II + compound extract, and the combined composition group. As shown in the figure, the relative expression level of IL-12 mRNA in the combined composition group was significantly higher than that in the other groups.

[0047] Dendritic cells (DCs) are key antigen-presenting cells that initiate immune rejection. This experiment demonstrates that the complete composition provided by this invention significantly inhibits DC maturation, downregulates co-stimulatory molecules CD80, CD86, and MHCII, thereby blocking the "first signal" of T cell activation; simultaneously, it reduces the secretion of the pro-inflammatory factor IL-12, thus inhibiting the Th1 immune response. This effect is attributed to the synergistic effect of peptide II's DC targeting and the anti-inflammatory properties of the compound extract. Notably, the CD80 reduction rate in the single peptide II group and the single traditional Chinese medicine group was lower than that in the composition group, indicating that the synergistic effect of the composition provided by this invention is superior to pairwise combinations, demonstrating the precise regulatory advantage of the composition in the immune initiation process.

[0048] Example 4: Verification Experiment 1. Test sample The seven groups of solutions are the same as in Example 1, namely: Peptide I group: 1.5 mg / mL peptide I solution; Peptide II group: 1.0 mg / mL peptide II solution; Compound extract group: 3.0 mg / mL compound extract solution; Peptide I + Peptide II group: 1.5 mg / mL peptide I + 1.0 mg / mL peptide II mixed solution; Peptide I + Compound extract group: 1.5 mg / mL peptide I + 3.0 mg / mL compound extract mixed solution; Peptide II + Compound extract group: 1.0 mg / mL peptide II + 3.0 mg / mL compound extract mixed solution; Composition group: 10 mg / mL composition solution of the present invention (including 1.5 mg / mL peptide I, 1.0 mg / mL peptide II, and 3.0 mg / mL compound extract).

[0049] 2. Test methods Using 96-well cell culture plates, 100 μL of autosensitized T cell suspension (cell density 1 × 10⁶ cells / well) was added to each well. 6 cells / mL, that is, 1×10⁻⁶ cells / mL per well 5(110 μL of cells), then add 10 μL of the corresponding sample solution (total volume 110 μL), 6 replicates per group, culture for 48 hours, and set up a control group with an equal volume of physiological saline.

[0050] The expression levels of TNF-α and IL-6 were detected by ELISA (kit: R&DSystems). A control group was also included, with an equal volume of physiological saline added. The reduction rates of TNF-α and IL-6 expression were calculated, and these rates were the percentage changes relative to the control group.

[0051] The proportion of Treg cells was detected by flow cytometry. A control group was included, which received an equal volume of physiological saline. The rate of increase in the proportion of Treg cells was calculated, and this rate of increase was the rate of change relative to the control group.

[0052] 3. Experimental Data and Results like Figure 6 The figures show the TNF-α reduction rates of the peptide I group, peptide II group, compound extract group, peptide I + peptide II group, peptide I + compound extract group, peptide II + compound extract group, and combined group on autologous sensitized T cells. As shown in the figure, the TNF-α reduction rate of the combined group was significantly higher than that of the other groups.

[0053] like Figure 7 The figures show the IL-6 reduction rates of the peptide I group, peptide II group, compound extract group, peptide I + peptide II group, peptide I + compound extract group, peptide II + compound extract group, and combined group on autologous sensitized T cells. As shown in the figure, the IL-6 reduction rate of the combined group was significantly higher than that of the other groups.

[0054] like Figure 8 The figure shows the increase rate of Treg cell proportion after treatment of autosensitized T cells with peptide I group, peptide II group, compound extract group, peptide I + peptide II group, peptide I + compound extract group, peptide II + compound extract group, and combination group. As shown in the figure, the increase rate of Treg cell proportion in the combination group was significantly higher than that in the other groups.

[0055] Chronic rejection mediated by autosensitized T cells is a significant cause of stem cell transplantation failure. This experiment demonstrates that the complete composition provided by this invention addresses this problem through a dual mechanism: First, the composition potently inhibits pro-inflammatory factors TNF-α and IL-6, with significantly better inhibitory effects than the single-herb group, the combination of peptide I and herb, and the combination of peptide I and peptide II. This reflects the synergistic blocking effect of the three components on inflammatory pathways such as NF-κB. Second, the composition promotes Treg cell proliferation, thereby enhancing the specific inhibition of autosensitized T cells. This synergistic anti-inflammatory and regulatory effect of the composition provided by this invention offers a more comprehensive solution for reducing chronic transplant rejection.

[0056] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A composition for reducing stem cell transplant rejection, wherein the reduction of stem cell transplant rejection is achieved by significantly inhibiting DC maturation, downregulating co-stimulatory molecules CD80, CD86, and MHCII, thereby blocking the "first signal" for T cell activation, and reducing the secretion of the pro-inflammatory factor IL-12, thereby inhibiting the Th1 immune response, characterized in that, The composition comprises peptide I: Val-Glu-Ser-Thr-Pro-Arg-Ala-Gly, peptide II: Leu-Asn-Gly-Lys-Met-Glu-Ser-His, and a compound extract; the compound extract is composed of Sinomenium acutum, Paeonia lactiflora, and Angelica sinensis in a mass ratio of 3:2:1, wherein the average weight content of Sinomenium acutum is ≥15%, the average weight content of paeoniflorin is ≥10%, and the average weight content of ferulic acid is ≥8%.

2. The composition according to claim 1, characterized in that, The composition comprises, by weight, 10-20 parts of peptide I, 5-15 parts of peptide II, and 20-40 parts of compound extract.

3. The composition according to claim 1, characterized in that, The composition comprises, by weight, 15 parts of peptide I, 10 parts of peptide II, and 30 parts of compound extract.

4. The composition according to claim 1, characterized in that, The composition contains 1.5 mg / mL peptide I, 1.0 mg / mL peptide II, and 3.0 mg / mL compound extract.

5. The use of the composition according to any one of claims 1 to 4 in the preparation of an agent that reduces stem cell transplant rejection.

6. The use of the composition according to any one of claims 1 to 4 in the preparation of formulations that directly inhibit effector T cell activation, induce immune tolerance, and regulate immune homeostasis.

7. The use of the composition according to any one of claims 1 to 4 in the preparation of an agent that downregulates co-stimulatory molecules and reduces the secretion of pro-inflammatory factor IL-12, wherein the co-stimulatory molecules are CD80, CD86 and MHCII.

8. The use of the composition according to any one of claims 1 to 4 in the preparation of an agent that inhibits the expression of pro-inflammatory factors TNF-α and IL-6 and promotes the proliferation of Treg cells.

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