Aplastic Anemia (AA) is a syndrome of bone marrow hematopoietic failure manifested also by pancytopenia, hemorrhage, and infection. The pathogenesis of AA involves Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs), which are key components of the bone marrow hematopoietic microenvironment and hematopoietic regulation. Abnormal activation of the immune system plays an important role in the pathogenesis of aplastic anemia. The immunosuppressant Cyclosporine (CsA) is widely used to treat AA, but its effect on BM-MSCs remains unclear. In this study, the effects of CsA on proliferation, apoptosis, morphology, matrilineage differentiation and immune regulation of BM-MSCs in AA patients were verified. Our results show that CsA regulates the bone marrow microenvironment in AA by reducing BM-MSCs adipogenic differentiation through the transforming growth factor beta 1 signaling pathway. Meanwhile, CsA can upregulate the expression levels of PD-L1 and PD-L2, especially PD-L1 positive cells, by transforming growth factor beta secretion and/or reducing the levels of IL6, further enhance the immunosuppressive ability of BM-MSCs which is beneficial for treating AA.

• Cyclosporin A

• Aplastic Anemia

• Mesenchymal Stem Cell

• Immunological Regulation

• Signaling Pathway

• Programmed Cell Death Protein 1

Shi LF, Sun L, Ma BD, Guo ZQ, Jin RR, et al. (2024) Cyclosporin a Inhibits Adipogenic Differentiation through the TGF- β1 Signaling Pathway and Regulates Immunomodulatory Functions of Mesenchymal Stem Cells in Aplastic Anemia Patients. Arch Stem Cell Res 6(1): 1020.

Aplastic Anemia (AA) is a syndrome affecting hematopoietic Stem Cells and their microenvironment and is associated with bone marrow tissue lipogenesis, abnormal function, and pancytopenia [1] as well as infection and hemorrhage. The latter clinical manifestations are mediated by aberrant immune cells [2]. Which directly and indirectly destroy stem/progenitor cells in the bone marrow by secreting gamma interferon, tumor necrosis factor alpha, and interleukins. Meanwhile, hematopoiesis is suppressed as a result of decreased production of early hematopoietic growth factors by stromal cells [3]. In addition to the sharp decline in the number of hematopoietic cells in the bone marrow of patients with AA, there is a dramatic increase in the number fat cells [4]. As lipogenesis is a notable pathological feature of AA, it is possible that this is related to abnormal differentiation of stem cells in bone marrow.

Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) self-renew, proliferate, and differentiate into a variety of cell types, including neurons, glia, and endothelial cells, as well as adipose and cartilage [5,6] and have therefore attracted considerable attention in the field of tissue regeneration. MSCs help maintain hemopoiesis and exert immune regulation, but are altered in patients with a variety of hematological diseases. BM-MSCs from patients with AA have enhanced angiogenesis as well as intrinsic deficits that contribute to their vulnerability to bone marrow failure. In addition, BM-MSCs have negative immunomodulatory properties, secreting cytokines to regulate T and B-lymphocytes, NK cells, and dendritic cells. MSCs express cell surface molecules with an immunosuppressive capacity, such as Programmed Death Ligand 1 (PD-L1) [7].

Lipogenesis is one of the most significant pathological features of bone marrow in aplastic anemia patients. The number of hematopoietic cells in the bone marrow of aplastic anemia patients decreases sharply while the number of fat cells increases significantly [8]. Obviously, these fat cells originate from the lipogenesis of bone marrow cells. Therefore, we speculated that the advocates in the bone marrow cavity of aplastic anemia patients might be related to the abnormal differentiation potential of MSC in aplastic anemia patients.

Current standard regimens for acquired AA include antilymphocyte/antithymocyte globulin and cyclosporine (or Cyclosporin A [CsA]), which target overactivated T cells [9,10], or marrow transplantation. Antilymphocyte/antithymocyte globulin selectively binds T lymphocytes and, with serum complement, lyses peripheral blood lymphocytes and reduces the expression of Fas antigen in CD34 + cells in bone marrow. At the same time, antithymocyte globulin also promotes the secretion of hematopoietic cell growth factor from lymphocytes. CsA, an immunosuppressant that is commonly used to treat organ transplant rejection and autoimmune disease as well as AA [11,12], inhibits the production of IL-1 by macrophages and the expression of the IL-1 receptor on Th cells and indirectly enhances the growth of granular monocytes. However, the effect of CsA on human BM-MSCs is not known. The objective of this study was to assess whether CsA affects proliferation, apoptosis, and immunomodulatory functions of human BM-MSCs. Furthermore, this study explored the role of CsA in lipogenesis as a novel mechanism for the therapeutic effects of CsA on BM-MSCs in patients with AA.

Culture of Human BM-Mscs

Three patients diagnosed with AA according to the criteria of The Fourth National Aplastic Anemia Conference (1987) at the blood department of Zhengzhou Central Hospital affiliated to Zhengzhou University were included in the study. Three marrow samples were obtained from the ribs that were removed from bone marrow puncture. Patients were informed of the content of the study and voluntarily acceded to participate. All donors provided written informed consent, and the Ethics Committee of Zhengzhou Central Hospital Affiliated to Zhengzhou University approved the present study. Those who had recently received related drug treatment, had contraindications to CsA, autoimmune diseases, or malignant tumors were excluded from the study.

BM-MSCs were extracted from patient samples named AA- BM-MSCs and cultured in Dulbecco’s modified Eagle medium/ F12 supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Only BM-MSCs passaged 1 to 3 times were used in the study. When cells reached semi confluence, culture plates were washed and the MSCs phenotype confirmed by flow cytometry (Cytoflex, Beckman Coulter) using the specific antibodies anti-CD73-PE, anti-CD90-APC, anti- CD105-APC-A750, anti-CD34, anti-CD45, anti-CD11b, anti-CD19, and anti-HLA-DR-FITC (Biolegend, San Diego, CA USA).

Cell Proliferation

Here AA-BM-MSCs were treated with the drug on two groups, which respectively named CsA and NC (DMSO negative control). AA-BM-MSCs were seeded at a density of 7×103 cells per well in 96-well culture plates and treated with CsA (Novartis Pharma AG, Basel, Switzerland) at concentrations of 0, 0.5, 1, 2, 5, and 10 μg/ ml for 24 h. Cell proliferation was measured by the CCK8 assay (Beyotime, Shanghai, China), and the absorbance was measured at 450 nm using a microplate reader (BioTek Instruments, Inc. Winooski, VT, USA).

Cell Apoptosis

AA-BM-MSCs were plated at a density of 7×104 cells per well in 6-well culture plates and treated with CsA (0-10 μg/ml) for 24 h. Apoptosis was assessed by staining with annexin V/fluorescein isothiocyanate and propidium iodide and analyzed using a Beckman flow cytometer (Beckman Coulter, Brea, CA, USA).

Flow Cytometry Analysis

Cells were resuspended in 500 μl PBS with antibodies against anti-CD73-PE, anti-CD90-APC, anti-CD105-APC-A750, anti-CD34, anti-CD45, anti-CD11b, anti-CD19, anti-HLA-DR-FITC, anti-PDL1- FITC, and anti-PDL2-PE (all from BioLegend, USA) at 4°C for 30 min. The samples were washed twice, and analyzed using a Beckman flow cytometer (Beckman Coulter, Pasadena, CA).

AA-BM-MSCs Adipocyte Differentiation and Osteogenic Differentiation

To induce adipocyte or osteogenic differentiation, AA-BM- MSCs were plated at a density of 3×104 cells per well in 6-well plates and treated with CsA at 2 μg/mL for 72 h (we found CsA almost had no effect on cell proliferation and apoptosis at this concentration). For some experiments, cells were pretreated with 5 nM A8301 (an inhibitor of transforming growth factor beta 1 receptor kinases ALK4, -5, and -7) for 1 h before CsA was applied. Osteogenic differentiation medium (Lonza) containing L-glutamine, mesenchymal cell growth serum MCGS, dexamethasone, ascorbate, β-glycerophosphate, and pen/strep. Adipogenic induction medium (Lonza) containing additional h-insulin, L-glutamine, MCGS, dexamethasone, indomethacin, 3-isobuty-1-methyl-xanthine, and pen/strep. Cell culture medium was changed every 2-3 days for a total of 21 days. On day 21, Oil Red O solution was used to stain the accumulated lipid droplets in differentiated adipocytes observed by microscopy. Alizarin red staining (Solarbio, Beijing, China) was performed to assess osteogenic differentiation.

RNA Isolation and qPCR

Total RNA was isolated from AA-BM-MSCs for real-time PCR (qPCR) using the primers listed in (Table 1). Gene expression was normalized to β-actin levels.

Table 1: Primer sequences used in this study.

Enzyme-Linked Immunosorbent Assay (ELISA)

AA-BM-MSCs were stimulated with CsA at 2 μg/ml for 72 h. Secretion of transforming growth factor beta (TGF-β) was measured using ELISA as per the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA).

Flow High Flux Multifactor Detection

The supernatants of AA-BM-MSCs+CsA co-cultured for 24 h were collected and Legendplex TM human Th cytokine assay (Biolegend) was used to analyze the following 13 factors: IL-2, 4, 5, 6, 9, 10, 13, 17A17F, 21, 22, IFN-γ and TNF-α. Experiments were repeated to ensure the accuracy of the data. Finally, Cytoflex flow cytometer (Beckman Coulter, Krefeld, USA) was used for analysis and Legendplex V8.0 and Prism7.0 software (Biolegend) analyzed the data, the concentration was ng/ml.

Statistical Analysis

Statistical analyses were performed using Prism 7.0 (GraphPad Software, San Diego, CA) SPSS professional statistical software. The experimental results are reported as means ± standard deviations. The Student t test was used to compare two groups, and one-way analysis of variance was used for comparisons among groups. A P value of < 0.05 was considered statistically significant.

CsA Decreased AA-BM-Mscs Proliferation and Increased Cell Apoptosis

To investigate whether CsA affects AA-BM-MSC proliferation and apoptosis, cells were treated with CsA at concentrations of 0, 0.5, 1, 2, 5, and 10 μg/ml for 24 h. CCK-8 assays showed that CsA significantly inhibited AA-BM-MSC proliferation at 5 μg/ml and 10 μg/ml (Figure 1A).

Figure 1: Effects of CsA on the proliferation and apoptosis of AA-BM-MSCs.

A. Cell proliferation of BM-MSCs treated with CsA (0-10 μg/ml) was assayed by CCK8.

B. The mRNA expression of BCL2 and BAX in BM-MSCs was determined by real-time PCR.

C,D. CsA promoted cell apoptosis in vitro according to annexin V-FITC/propidium iodide double staining and flow cytometry. ***P < 0.001.

This concentration also reduced the ratio of antiapoptotic Bcl-2 gene expression to that of proapoptotic Bax according to qPCR (Figure 1B). Accordingly, annexin V/ fluorescein isothiocyanate and propidium iodide staining revealed that 5 μg/ml and 10 μg/ml CsA increased the percentage of cells undergoing apoptosis (Figure 1C,D). Thus, CsA promotes both early and late phases of apoptosis.

CsA Had No Effect on Immunophenotype and Osteogenic Differentiation of AA-BM-Mscs

To determine whether CsA alters the immunophenotype of AA-BM-MSCs, the expression of key surface markers was tested by flow cytometry, such as (Figure 2A).

Figure 2: Effects of CsA on surface markers and osteogenic differentiation of AA-BM-MSCs.

A. Flow cytometry for surface markers of human BM-MSCs treated or not with 2 μg/ml CsA. The histogram shows the relative expression of each factor.

B. The osteogenic mRNA expression of Ocn, Col1a1 and Runx2 was determined in 3d, 13d and 21d.

C. Osteogenic differentiation of BM-MSCs was illustrated by Alizarin red staining

However, the expression of these cell-surface markers was unaffected by treatment for 72 h with 2 μg/ml CsA. After osteogenic induction for 3, 13 and 21 days, the osteogenic gene expression and Alizarin red staining in BM-MSCs were observed. We found that CsA did not affect the mRNA expression of OCN, COL1a1 and Runx2 at 2 μg/ml (Figure 2B). Three weeks after osteogenic induction, we found no evidence that CsA could promote the osteogenic differentiation of AA-BM-MSCs, as illustrated by Alizarin red staining (Figure 2C).

CsA Inhibited The Lipogenic Differentiation Of AA- BM-Mscs Through The TGF- β1 Signaling Pathway

AA-BM-MSCs were treated with CsA for 3 days at 2 μg/ml and were cultured in appropriate conditions for inducing the adipogenic differentiation. Our results indicated that the mRNA expressions of PPAR-γ was significantly decreased in CsA-treated AA-BM-MSCs (Figure 3A).

Figure 3: Effect of CsA on lipogenic differentiation and TGF-β concentrations of AA-BM-MSCs.

A,D. Oil-Red O stainings of CsA-treated, CsA+A8301-treated AA-BM-MSCs and controls after cultured in adipogenic medium for 21 days.

B,E. The mRNA expression of adipogenic PPAR-γ was determined by real time PCR.

C. TGF-β concentrations were measured by enzyme-linked immunosorbent assays.***P < 0.001.

Oil Red-O staining results showed that CsA inhibited the lipogenic differentiation of AA-BM-MSCs (Figure 3B). It is widely assumed that TGF-β exerts an inhibitory action on human adipose tissue. Then we analyzed TGF-β secretion by ELISA. Our results showed that the secretion level of TGF-β in the supernatant was considerably increased with CsA treatment (Figure 3C).

To further confirm the inhibitory effect of CsA on the adipogenic differentiation of AA-BM-MSCs, we pretreated the cells with TGF-β1 signaling pathway block-blocking agent A8301 (5 nM), and detected the lipid differentiation ability, expression level of mRNA of PPAR-γ. Our results showed that lipotropic ability was significantly up regulated after induction by the lipotropic medium (Figure 3D). At the same time, the mRNA expression level of PPAR-γ was also significantly up-regulated (Figure 3E). These results suggest that CsA mainly inhibits the lipogenic differentiation of AA-BM-MSCs in vitro through the TGF-β1 signaling pathway.

CsA Treatment Inhibited AA-BM-Mscs Secreting IL-6 And Upregulated The Expression Of PD-L1 And PD-L2

As we know, CsA is an immunosuppressant that selectively acts on the CD4 + subset of T lymphocytes. So we determine the effect of CsA on the immunomodulation function of AA-BM-MSCs, we analyzed cytokine production via flow high-flux multifactor detection. This panel allows simultaneous quantification of 13 human cytokines, including IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21,22, IFN-γ and TNF-α, which are collectively secreted by Th1, Th2, Th9, Th17, and Th22. Medium from the CsA-treated AA-BM-MSCs had lower concentrations of IL-13, and IFN-γ than NC, and the secretion level of IL-6 was significantly reduced (Figure 4A).

Figure 4: Effect of CsA on the immunomodulation function of AA-BM-MSCs.

A. IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 21, 22, IFN-γ and TNF-α were detected by Human the Cytokine Panel.

B. mRNA expression levels of PD-L1 and PD-L2 in human AA-BM-MSCs treated or not with CsA.

C. Surface expression of PD-L1 were assessed by flow cytometry.

D. Quantitative analysis of PD-L1 positive cells from three independent experiments.

E. Surface expression of PD-L2 were assessed by flow cytometry.

F. Quantitative analysis of PD-L2 positive cells from three independent experiments.*P < 0.05, ***P < 0.001.

As previously reported, TGF-β1 and IL-6 signaling can regulate the expression of PD-L1 and PD-L2 [13,14]. Therefore, we verified the expression of PD-L1 and PD-L2. CsA treatment upregulated the expression of PD-L1 and PD-L2 mRNA in AA- BM-MSCs (Figure 4B). Flow cytometry analyses revealed that protein expression of PD-L1 and PD-L2 on CsA-treated AA-BM- MSCs was all higher than controls (Figure 4C,E). Interestingly, almost all (95.74% ± 3.25%) AA-BM-MSCs expressed PD-L2 (Figure 4F). However, CsA treatment increased the percentage of PD-L1 positive cells from 9.98% ± 2.47% to 58.32% ± 2.85% (Figure 4D).

Cyclosporine (CsA) is a traditional immunosuppressant that is commonly used to treat organ transplant rejection, Aplastic Anemia (AA) and autoimmune diseases [15,16]. BM-MSCs are the main component of the bone marrow microenvironment.

The main function of MSCs is to maintain hemopoiesis and exert immune regulation [17,18]. But they can also differentiate into adipose, osteogenic, and cartilage tissues and secrete a variety of factors. MSCs are critically altered in patients with different hematological diseases [19,20]. For example, BM-MSCs from patients with AA enhance angiogenesis, which may play a role in the pathogenesis of the disease [21]. Qu, et al [22] showed that the proliferation and adipogenic differentiation of murine BM-MSCs are inhibited by CsA, which also promotes their apoptosis. Here, we show that CsA affects the proliferation, apoptosis, phenotype, multidirectional differentiation ability, and cytokine secretion of human BM-MSCs from AA patients.

In our study, we confirmed that CSA inhibits BM-MSC proliferation and promotes apoptosis in a dose-dependent manner. Although the mechanism behind the ant proliferative effect remains unclear, CsA has been shown to impair proliferation and osteoclast differentiation via the nitric oxide synthase pathway [23]. CsA-mediated apoptosis, however, likely involved the decrease in the ratio of Bcl-2, one of the most important regulators of apoptosis in mammalian cells, to Bax, a molecule that initiates mitochondrial apoptosis pathways [24,25]. This suggests that Bax and Bcl-2 are involved in CsA inducing apoptosis of BM-MSCs.

Surface antigen expression of MSCs includes (CD73, CD90 and CD105). BM-MSCs have no or low expression of co- stimulatory molecules CD19, CD34, CD45, CD11b and HLA-DR, which contribute to low immunogenicity of BM-MSCs. In our study, we found that CsA did not affect the immunophenotype of BM-MSCs. Fatty Bone Marrow (BM) and defective hematopoiesis are a pathologic hallmark of Aplastic Anemia (AA). Existing literature reports that BM-MSC of AA patients had a morphology, phenotype, and osteogenic differentiation potential similar to normal people but adiposity differentiated from AA BM-MSC had a higher density and larger size of lipid droplets and they expressed significantly higher levels of adiponectin and FABP4 genes and proteins as compared to control BM-MSC [26,27]. AA-BM-MSC have enhanced angiogenesis, which may have an important implication in the pathogenesis of the disease. These studies indicate that BM-MSCs may be associated with the pathogenesis of AA through regulating the microenvironment and inhibiting adipogenesis may be a new therapeutic method for AA. In this study, we investigated the effect of CsA on adipogenic and osteogenic differentiation in BM-MSCs.

In addition, we also examined the lipogenic and osteogenic genes of bone marrow mesenchyme stem cells in aplastic anemia patients with or without CsA treatment CsA inhibited BM-MSCs adipogenic differentiation obviously, while no significant effect on osteogenic differentiation. At the same time, we investigated the changes of cytokines and found that the secretion level of TGF-β in BM-MSCs significantly increased after CsA treatment. We speculated whether CsA was involved in the lipid differentiation of BM-MSCs through the TGF-β pathway. In addition, Gilson [28,29] pointed out that the decreased concentration of TGF-β1 in the blood circulation of aplastic anemia patients was significantly correlated with the occurrence of aplastic anemia. TGF-β1 is an important regulator involved in the adipogenic differentiation of BM-MSCs, and its specific regulatory mechanism remains unclear. In recent years, some progress has been made in its research. Tsurutani, et al [30] reported that TGF-β plays an important role in regulating adipose differentiation of mouse embryonic fibroblasts, but the mechanism is not clear. Mu, et al [31] found that TGF-β can exert multiple biological effects in stem cells through Smad signaling pathways and non-smad signaling pathways. Then we did the TGF-β1 pathway inhibition test. Our results showed that lipotropic ability was significantly up- regulated after induction by the lipotropic medium. At the same time, the mRNA expression level of PPAR-γ was also significantly up-regulated. These results showed that CsA could regulate the bone marrow microenvironment by affecting BM-MSCs through the TGF-β1 signaling pathway, thus promoting the treatment of AA.

Several soluble factors produced by MSCs have been reported to mediate the suppression of T-cell proliferation [32]. IL-6 is a cytokine synthesized and secreted by mononuclear phagocytes and vascular endothelial cells, which are involved in the regulation of immune system function and hematopoietic function of bone marrow tissue [33,34]. Gupta et al found that IL-6 is involved in the severity of children with aplastic anemia [35]. Earlier studies found that IL-6 aggravates the condition of patients with aplastic anemia [36]. The application of immunosuppressive agents at high levels of IL-6 in children with aplastic anemia is effective [37]. However, whether it has a role in aplastic anemia needs further research. Reported that CsA acts selectively on T lymphocyte subsets. Namely, it inhibits the activation and proliferation of T cells by blocking expression of the IL-2 receptor and suppressing the production of IL-2 and IFN-γ. Our study confirmed that CsA may inhibit the secretion of IL-6, IL-13 and IFN-γ by AA bone marrow mesenchyme stem cells to regulate the immune function of AA patients. And then achieve the effect of treating AA. Our results suggested that CsA can inhibit T cell activation, also can improve the bone marrow microenvironment of AA by acting on BM-MSCs. This is consistent with Qu, et al [22] research in murine BM-MSCs.

Previous study showed that BM-MSCs inhibited lymphocyte proliferation via engagement of the inhibitory molecule programmed death-1 (PD-1) to its ligands PD-L1 and PD-L2 [38- 40]. Fresh reports of aplastic anemia as immune-related adverse events (irAEs) implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche [41]. David JM, et al [42] showed that TGF-β was found to up-regulate PD-L1 gene transcription in a Smad2-dependent manner in non-small cell lung cancer cells. Our study discovered that CsA may significantly promote the secretion of TGF-β1 by AA bone marrow mesenchyme stem cells. These findings demonstrate that up-regulation of BM-MSCs PD-L1 is a novel mechanism of TGF-β-induced immunosuppression in CsA treated AA.

We also found CsA significantly inhibits the secretion of IL-6 by AA bone marrow mesenchyme stem cells. Gupta, et al found that IL-6 is involved in the severity of children with aplastic anemia [42]. Earlier studies found that IL-6 aggravates the condition of patients with aplastic anemia. The application of immunosuppressive agents at high levels of IL-6 in children with aplastic anemia is effective [37]. Mace, Hirotake T, et al [14] showed that targeted inhibition of IL-6 may enhance the efficacy of anti-PD-L1 in pancreatic ductal adenocarcinoma. Fang Xu, et al [14] showed that PD-L1 Mesenchymal Stem Cell (MSC) derived extracellular vesicles (MSCs-EVs-PD-L1) exhibits an impressive ability to regulate various activated immune cells to an immunosuppressed state in vitro. Hence, our results indicate a novel mechanism for CsA influencing BM-MSCs. In the treatment of AA with CsA, blocking the IL-6 pathway can help to up-regulate the expression of PD-L1 and enhance the immunosuppressive effect of BM-MSCs to enhance therapeutic effect.

Our date showed that CsA inhibited AA-BM-MSC proliferation, while promoting AA-BM-MSCs apoptosis. In addition, CsA induced adipogenic differentiation, but had no effect on osteogenic differentiation in AA-BM-MSCs. We found that higer concentration of TGF-β was detected in the CsA-treated AA-BM- MSCs medium than NC. In addition, we also found that CsA could regulate the bone marrow microenvironment by affecting AA-BM- MSCs adipogenic differentiation through the TGF-β1 signaling pathway. Thus, in the treatment of AA with CsA, blockade of the IL-6 pathway may facilitate PD-L1 expression in AA-BM-MSCs to enhance the therapeutic immunosuppressive effect, which promote the treatment of AA. According to the above results, we will pay attention to the changes of the immune regulatory function of the AA-BM-MSCs on T cell subsets in patients with highly expressed PD-L1 aplastic anemia in the future work.

All listed authors have made substantial contributions to the following aspects of the manuscript:

1. The conception and design of the study, or acquisition of data, or analysis and interpretation of data.
2. Drafting the article or revising it critically for important intellectual content.
3. Final approval of the version to be submitted.

Ethics Approval and Consent to Participate: Not applicable.

Human and Animal Rights: No Animals/Humans were used for studies that are base of this research.

Consent for Publication: Not applicable.

Availability of Data and Materials: The data that support the findings of this study are available on request from the corresponding author.

Funding: This work was supported by Henan Province science and technology research and development projects (grant numbers 222102310032).This work was also supported by the Medical science and Technology project of Henan Province (grant numbers LHGJ20200764).This work was also supported by the Medical science and Technology project of Henan Province (grant numbers LHGJ20210775).

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