Immune Thrombocytopenia Patients Research Articles

Platelet-derived TGF-β1 induces functional reprogramming of myeloid-derived suppressor cells in immune thrombocytopenia

Platelet α-granules are rich in TGF-β1 which is associated with myeloid-derived suppressor cell (MDSC) biology. Responders to thrombopoietin receptor agonists (TPO-RAs) revealed a parallel increase in the number of both platelets and MDSCs. Here, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to establish an active murine model of immune thrombocytopenia (ITP). Subsequently, we demonstrated that TPO-RAs augmented the inhibitory activities of MDSCs by arresting plasma cells differentiation, reducing Fas ligand expression on cytotoxic T cells, and re-balancing T cell subsets. Mechanistically, transcriptome analysis confirmed the participation of TGF-β/Smad pathways in TPO-RA-corrected-MDSCs, which was offset by Smad2/3 knockdown. In platelet TGF-β1-deficient mice, TPO-RA-induced amplification and enhanced suppressive capacity of MDSCs was waived. Furthermore, our retrospective data revealed that ITP patients achieving complete platelet response showed superior long-term outcomes compared with those who only reach partial response. In conclusion, we demonstrate that platelet TGF-β1 induces the expansion and functional reprogramming of MDSCs via the TGF-β/Smad pathway. These data indicate that platelet recovery not only serves as an endpoint of treatment response, but also paves the way for immune homeostasis in immune-mediated thrombocytopenia.

The evolution of preexisting primary immune thrombocytopenia after COVID-19 onset: A nationally representative, prospective, multicentre, observational study.

The symptoms in patients with primary immune thrombocytopenia (ITP) after COVID-19 onset remain largely unclear. The aim of this study was to describe the platelet count fluctuations in ITP patients following the diagnosis of COVID-19. A prospective multicentre observational study was conducted from December 15th, 2022, to January 31st, 2023 in 39 general hospitals across China. Patients with preexisting primary ITP who were newly diagnosed with COVID-19 were enrolled. A total of 1216 ITP patients with newly-diagnosed COVID-19 were enrolled. 375 (30.8%) patients experienced ITP exacerbation within eight weeks after the diagnosis of COVID-19, and most exacerbation (266/375, 70.9%) developed in the first two weeks. Immunosuppressive therapy for ITP and severe/critical COVID-19 infection were independent variables associated with ITP exacerbation. Overall the platelet count had a transient increasing trend, and the platelet peak value occurred at two weeks after COVID-19 infection. Then, the platelet count decreased to the baseline level in the following weeks. The platelet count had a transient increasing trend in ITP patients following the diagnosis of COVID-19. ITP exacerbation only occurred in less than one-third of ITP patients. Nonimmunosuppressive therapy may have an advantage to prevent ITP exacerbation during COVID-19.

Comparison of platelet transfusion effectiveness between Helicobacter pylori-positive and -negative immune thrombocytopenia.

Immune thrombocytopenia (ITP) is characterized by early platelet destruction and impaired platelet production. Helicobacter pylori (H. pylori) infection seems to contribute to the pathogenesis in certain ITP patients in Japan. We compared the effectiveness of platelet transfusion in severe ITP in the presence or absence of H.pylori. The median corrected count increment (CCI) at 24 h after platelet transfusion (CCI-24) of the H.pylori-positive ITP patients was higher than that of the H. pylori-negative ITP patients (6463 vs. 754, p < 0.001), and the CCI-1 was also in the same direction but not significant (23 351 vs. 11 578). Multiple regression analyses showed that H. pylori infection was independently associated with CCI-24. Our study suggests that platelet transfusion may be more effective in H. pylori-positive ITP patients.

Challenges in treating primary immune thrombocytopenia patients undergoing COVID-19 vaccination: A retrospective study.

Since the outbreak of COVID-19 in December 2019, countries around the world, including China, have been administering COVID-19 vaccines in response to the pandemic. Our center has observed that treating patients with primary immune thrombocytopenia (ITP) has become more challenging in this context. This study compared the treatment response of 25 de novo ITP patients who had received a COVID-19 vaccination (Group 1) with an equal number of de novo ITP patients randomly selected from the 2 years prior to the COVID-19 pandemic (Group 2) by using the Mann-Whitney U test and Fisher's exact. Patients in both groups had predominantly female gender with similar age and baseline platelet counts. However, on Day 3, the median platelets were 22 and 49 × 109/L, and on Day 7, they were 74 and 159 × 109/L, respectively (P < 0.05). Compared to Group 2, Group 1 showed a suboptimal short-term response to glucocorticoid monotherapy, with a higher proportion of patients requiring combination therapy with other drugs including intravenous immunoglobulin, thrombopoietin receptor agonists, and rituximab. After subgroup analysis, a significant difference was observed in the proportion of patients requiring second-line therapy between the two groups. Our study suggests that COVID-19 vaccination may lead to a lower response rate to first-line treatment in de novo ITP patients. Nevertheless, it is crucial to acknowledge the inherent limitations in this conclusion. Further studies are needed to confirm these findings and investigate the underlying mechanisms.

Evaluation of vitamin D status in adult patients with newly diagnosed immune thrombocytopenia.

It has been shown that 25-OH vitamin D not only preserves calcium and bone homeostasis but also has immunomodulatory effects. The purpose of this study was to assess the association between adult patients with recently diagnosed immune thrombocytopenia (ITP) and vitamin D levels. Retrospective technique was employed in this study. The associations between 25(OH)D value and platelet count, as well as the clinical symptoms of ITP upon diagnosis and 25(OH)D value, were our main findings. A total of 60 patients diagnosed and followed up in our clinic were included in the study. Forty-one patients (68.3%) were female and 19 (31.7%) were male. The median age of the patients was 52.5 (19-88). The median vitamin D level of all patients at diagnosis of ITP was 11.5 (3-86). There was no statistically significant difference between the patients divided into three groups according to their vitamin D levels, in terms of laboratory parameters. There was no statistically significant difference in clinical findings according to vitamin D status in ITP patients. There was no statistically significant difference in terms of relapse-free survival in all three groups (p = 0.71). In conclusion, in our study, no correlation was found between laboratory and clinical findings at diagnosis and vitamin D levels in adult ITP patients. Additional investigations, particularly randomized controlled trials, are required to examine the relationship between 25(OH)D and the incidence and severity of ITP.

Metabolomics profile and machine learning prediction of treatment responses in immune thrombocytopenia: A prospective cohort study.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by antibody-mediated platelet destruction and impaired platelet production. The mechanisms underlying ITP and biomarkers predicting the response of drug treatments are elusive. We performed a metabolomic profiling of bone marrow biopsy samples collected from ITP patients admission in a prospective study of the National Longitudinal Cohort of Hematological Diseases. Machine learning algorithms were conducted to discover novel biomarkers to predict ITP patient treatment responses. From the bone marrow biopsies of 91 ITP patients, we quantified a total of 4494 metabolites, including 1456 metabolites in the positive mode and 3038 metabolites in the negative mode. Metabolic patterns varied significantly between groups of newly diagnosed and chronic ITP, with a total of 876 differential metabolites involved in 181 unique metabolic pathways. Enrichment factors and p-values revealed the top metabolically enriched pathways to be sphingolipid metabolism, the sphingolipid signalling pathway, ubiquinone and other terpenoid-quinone biosynthesis, thiamine metabolism, tryptophan metabolism and cofactors biosynthesis, the phospholipase D signalling pathway and the phosphatidylinositol signalling system. Based on patient responses to five treatment options, we screened several metabolites using the Boruta algorithm and ranked their importance using the random forest algorithm. Lipids and their metabolism, including long-chain fatty acids, oxidized lipids, glycerophospholipids, phosphatidylcholine and phosphatidylethanolamine biosynthesis, helped differentiate drug treatment responses. In conclusion, this study revealed metabolic alterations associated with ITP in bone marrow supernatants and a potential biomarker predicting the response to ITP.

Effectiveness and safety of eltrombopag in connective tissue disease patients with refractory immune thrombocytopenia: a retrospective study.

We aimed to investigate the safety and effectiveness of eltrombopag for adult patients with refractory immune thrombocytopenia (ITP) secondary to connective tissue disease (CTD). This is a single-centre, retrospective cohort and propensity score-matched study. Data from CTD-ITP patients treated with eltrombopag between January 2019 and January 2023 were retrospectively analysed. Baseline characteristics and follow-up information were recorded. CTD patients without ITP were matched to identify the risk factors associated with CTD-ITP performed by Logistic regression analysis. Twenty patients were enrolled, including 5 systemic lupus erythematosus (SLE), 9 Sjögren's syndrome (SS) and 6 undifferentiated connective tissue disease (UCTD). Nineteen (95%) patients were female, and the median age was 59 years. Logistic regression analysis showed that anaemia (OR = 8.832, P = 0.007) was associated with increased risk of ITP, while non-erosive arthritis (OR = 0.045, P = 0.001) and interstitial lung disease (OR = 0.075, P = 0.031) were associated with reduced risk. Fourteen patients (70%) achieved a complete response (CR) and one (5%) achieved a partial response (PR). The median response time was 14 days. The median platelet count was 8.5 × 109/l at baseline of eltrombopag and increased to 122 × 109/l after 4 weeks. No adverse events were observed. Eltrombopag appears to be effective, safe and well-tolerated in refractory ITP patients with CTD; larger studies are needed to confirm the generalizability of these findings.

Rituximab versus Splenectomy in Chronic Primary ITP: Experience of a Single Hematology Clinic.

Immune thrombocytopenia (ITP) is an acquired immune-mediated disease that lacks an underlying etiology. Steroids are the main first-line treatment of ITP, while the second-line treatment consists primarily of splenectomy and rituximab. This study aimed to assess and compare the response to rituximab and splenectomy. This retrospective comparative study reviewed ITP patients treated at a single private hematology clinic from 2007 to 2019. Seventy-four ITP patients were recruited, 27 were on rituximab, and 47 had undergone splenectomy. The initial platelet counts and bleeding symptoms were recorded, and initial and long-term responses to treatment were evaluated based on the American Society of Hematology guidelines. The mean age of the patients was 42.1 years with a male-to-female ratio of 1:1.8. The initial mean platelet count was comparable between the rituximab and splenectomy groups (p = 0.749). The initial complete response (CR) differed significantly between the rituximab and splenectomy groups (44.4% versus 83%, p = 0.002). The five-year response rate was significantly higher in the splenectomy than in the rituximab group (74% versus 52%, log-rank 0.038). Splenectomy was the only significant predictive factor for long-term response (OR = 0.193, p = 0.006). The overall response revealed that splenectomy appeared superior to rituximab as a second-line treatment of ITP. Splenectomy was the only positive prognostic indicator of sustained response.

Изменения качества жизни у пациентов с хронической иммунной тромбоцитопенией в процессе терапии ромиплостимом, его эффективность и безопасность в условиях реальной клинической практики: результаты многоцентрового наблюдательного исследования

Aim. To study the quality of life in patients with chronic immune thrombocytopenia (ITP) in the process of romiplostim therapy and to assess the efficacy and safety of this drug in real-world setting.&#x0D; Materials &amp; Methods. The study enrolled adult patients with the confirmed chronic ITP diagnosis and indications for romiplostim therapy. Clinical parameters, RAND SF-36 and FACT-Th6 quality of life as well as FACIT-Fatigue scores were evaluated prior to romiplostim administration vs. 3, 6, and 12 months after the treatment onset. Patient satisfaction checklist was also administered at all study points after the start of therapy. The clinical efficacy of romiplostim was analyzed along with assessing response and time to response. To study the quality of life and fatigue changes, the Generalized Estimating Equation (GEE) method was used during the observation period. Significant fatigue changes were determined and compared in terms of the perception differences from patient’s and physician’s perspective.&#x0D; Results. The study enrolled 60 chronic ITP patients treated with romiplostim in the real-world setting (mean age 51.9 years, 70 % women). The median thrombocyte count prior to romiplostim therapy was 18.5 × 109/L (interquartile range 10.8–22.3 × 109/л). On the enrollment date, 90 % of patients showed hemorrhagic syndrome. Overall response to romiplostim therapy was 98.3 % (complete response was achieved in 93.3 % of patients). After 6 months of therapy, 89.5 % of patients preserved response. After 3 months of therapy, hemorrhagic syndrome was eliminated in 81 % of patients, after 6 months the same was achieved in 93 % of patients. The median time to response was 4.4 weeks (95% confidence interval 3.6–5.3 weeks). Adverse events of grades 1/2 associated with romiplostim were reported in 6.7 % of patients. On romiplostim therapy, pronounced positive changes in quality of life were shown by all scales of the general questionnaire SF-36 and the targeted questionnaire FACT-Th6 (p &lt; 0.001). The clearest improvements were observed in role-physical and role-emotional functioning. Already after 3 months of therapy, a considerable fatigue reduction was observed and sustained for the next 6 and 12 months of romiplostim administration (p &lt; 0.001). During the therapy, the proportion of patients with fatigue impacting various aspects of functioning became considerably smaller. The vast majority of patients (85 %) were satisfied with the treatment. Discrepancies between patients’ and physicians’ evaluations of fatigue were also identified during the treatment.&#x0D; Conclusion. The results of the present multi-center observational study demonstrate high efficacy and safety of romiplostim for chronic ITP patients in the real-world setting. Romiplostim therapy yields considerable quality of life improvement and fatigue reduction. To optimize the patient monitoring system and patient-centered ITP treatment in the real-world setting, it is advisable to use the standardized questionnaires assessing quality of life and fatigue.

GPIbα CAAR-T cells function like a Trojan horse to eliminate autoreactive B cells to treat immune thrombocytopenia.

Breakthrough treatment for refractory and relapsed immune thrombocytopenia (ITP) patients is urgently needed. Autoantibody-mediated platelet clearance and megakaryocyte dysfunction are important pathogenic mediators of ITP. Glycoprotein (GP) Ibα is a significant autoantigen found in ITP patients and is associated with poor response to standard immunosuppressive treatments. Here, we engineered human T cells to express a chimeric autoantibody receptor (CAAR) with GPIbα constructed into the ligand-binding domain fused to the CD8 transmembrane domain and CD3ζ-4-1BB signaling domains. We performed cytotoxicity assays to assess GPIbα CAAR-T-cell selective cytolysis of cells expressing anti-GPIbα B-cell receptors (BCRs) in vitro. Furthermore, we demonstrated the potential of GPIbα CAAR-T cells to persist and precisely eliminate GPIbα-specific B cells in vivo. In summary, we present a proof of concept for CAAR-T-cell therapy to eradicate autoimmune B cells while sparing healthy B cells with GPIbα CAAR-T cells that function like a Trojan horse. GPIbα CAAR-Tcell therapy is a promising treatment for refractory and relapsed ITP patients.

Is Immune Thrombocytopenia and its Treatment Associated with Sarcopenia?

Immune thrombocytopenia (ITP) is defined as an isolated platelet count less than 100 × 109/L in the absence of other causes of thrombocytopenia. Sarcopenia is a body-wide muscular disorder with a progressive nature that leads to reduced mobility, physical disability, falls, and poor quality of life. We aimed to evaluate the frequency of objectively diagnosed sarcopenia in patients with ITP and to determine whether ITP therapies have sarcopenic effects. This prospective study included patients who were followed up with ITP in the hematology outpatient clinic. Patients who had received corticosteroids within 3 months were excluded. The handgrip strength test, appendicular skeletal muscle mass (ASMM), ASMM/height2 value, soft lean mass (SLM), trunk soft lean mass (SLMT), and the 6-min walking speed test were applied for muscular evaluations and physical performance assessment. We included 53 patients (female/male: 73.58%/26.42%). While sarcopenia was not observed in 77.36% of ITP patients, possible sarcopenia was diagnosed in 9.43% and confirmed sarcopenia in 13.21%. Severe sarcopenia was not seen in any of the patients. Loss of muscle strength was observed in 22.64% of patients. SLM was found to be low in 92.45%. Sarcopenia may be more frequent among patients with ITP compared to the population, and it is important to note that 92.45% of patients had low SLM and 54.72% had low SLMT. Eltrombopag therapy might be beneficial as demonstrated by higher SLM, ASMM, and ASMM/height2 values.

Decreased Expression of IL-35 and Its Receptor Contributes to Impaired Megakaryopoiesis in the Pathogenesis of Immune Thrombocytopenia.

Recent findings have shown that the level of interleukin-35 (IL-35) is abnormal in several autoimmune diseases. Nonetheless, whether IL-35 participates in the pathogenesis of immune thrombocytopenia (ITP) remains unclear. The current study investigates whether IL-35 modulates megakaryopoiesis. The results show that IL-35 receptors are progressively expressed on bone marrow megakaryocytes during the in vitro differentiation of CD34+ progenitors. IL-35 increases the number of megakaryocyte colony-forming units through the Akt pathway. The level of bone marrow IL-35 is reduced in ITP patients, and the decreased level of IL-35 may inhibit megakaryopoiesis. Then, the potential causes of decreased IL-35 in ITP patients are explored. The primary type of cell that secretes IL-35, known as IL-35-producing regulatory T cells (iTr35), is reduced in ITP patients. Bone marrow mesenchymal stem cells (MSCs) from ITP patients exhibit an impaired capability of inducing iTr35 due to enhanced apoptosis, which may contribute to the reduced level of bone marrow IL-35 in ITP patients. Iguratimod promotes megakaryocyte development and differentiation by elevating the expression of IL-35 receptors on megakaryocytes. Iguratimod improves response rates and reduces bleeding symptoms in corticosteroid-resistant ITP patients.

Rectifying the impairment of immune thrombocytopenia plasmas through photobiomodulation

&lt;p&gt;Immune thrombocytopenia (ITP) is an autoimmune hemorrhage disorder. The first-line treatment of this disorder is corticosteroids, followed by thrombopoietin (TPO) receptor agonists such as Nplate, and/or splenectomy. Yet, the extended usage of corticosteroids or the expensive Nplate, coupled with the implications of splenectomy, raises concerns due to the array of associated side effects and an escalated vulnerability to subsequent complications. The current investigation shows that while anti-platelet antibodies and ITP plasmas hinder megakaryocyte differentiation and maturation and impair proplatelet and platelet formation in ex vivo culture of umbilical cord human CD34+ stem cells (cHSCs), low-level laser (LLL) treatment or photobiomodulation (PBM) effectively mitigates these detrimental impacts. PBM reinstated megakaryocyte differentiation and maturation, bolstering proplatelet and platelet formation in the presence of auto-platelet antibodies or ITP plasmas. The mitigating effects of PBM appear to pivot on its capacity to uphold cellular mitochondrial functionality and rectify the mitochondrial impairments engendered by anti-platelet antibodies or ITP plasmas. These findings underscore the potential of PBM as a safe and cost-efficient alternative for the management of a specific subset of ITP patients.&lt;/p&gt;

Rectifying the impairment of immune thrombocytopenia plasmas through photobiomodulation

&lt;p&gt;Immune thrombocytopenia (ITP) is an autoimmune hemorrhage disorder. The first-line treatment of this disorder is corticosteroids, followed by thrombopoietin (TPO) receptor agonists such as Nplate, and/or splenectomy. Yet, the extended usage of corticosteroids or the expensive Nplate, coupled with the implications of splenectomy, raises concerns due to the array of associated side effects and an escalated vulnerability to subsequent complications. The current investigation shows that while anti-platelet antibodies and ITP plasmas hinder megakaryocyte differentiation and maturation and impair proplatelet and platelet formation in ex vivo culture of umbilical cord human CD34+ stem cells (cHSCs), low-level laser (LLL) treatment or photobiomodulation (PBM) effectively mitigates these detrimental impacts. PBM reinstated megakaryocyte differentiation and maturation, bolstering proplatelet and platelet formation in the presence of auto-platelet antibodies or ITP plasmas. The mitigating effects of PBM appear to pivot on its capacity to uphold cellular mitochondrial functionality and rectify the mitochondrial impairments engendered by anti-platelet antibodies or ITP plasmas. These findings underscore the potential of PBM as a safe and cost-efficient alternative for the management of a specific subset of ITP patients.&lt;/p&gt;

Immune thrombocytopenia (ITP) with corticosteroids: “Evaluation of Response”

Objective: to evaluate the response of corticosteroids in newly persistent ITP patients. Methodology: Descriptive Case series conducted from 30th October 20202 to 29th April 2021 at Armed Forces Bone Marrow Transplant Centre Rawalpindi. A total of 74 individuals with ITP who were both males and females of various ages. Prednisolone was administered orally to patients in maximal dose of 1mg/kg. The initial reaction of platelets count was assessed on day 5 and subsequently every two weeks until the full response was attained. An informed consent was obtained. Results: Age was 32.0±19.1 years on average. Twenty-four (32.4%) of the patients were over the age of 40 while the majority (n=50, 67.6%) of the patients were under the age of 40 years. There were 1:1.1 patients that were male and female with 36 (48.6%) men and 38(51.4%) women. Twenty (27.0%) individuals had persistent ITP compared to 54 (73.0%) patients who had just been diagnosed. 34 (45.9%) patients had an initial response whereas 43 patients (58.1%) showed a full response. The average initial response time was 5.47±2.39 days and 11.79±6.73 days for full response. The frequency of first didn’t differ statistically significantly by patient age, gender or other subgroups. Conclusion: Regardless of age, gender, or ITP type, steroids were associated with an initial and complete response in a significant majority of patients with immune thrombocytopenia. Keeping in view with their affordability accessibility favors the use of steroids as first line in these patients. 

Distribution of peripheral blood cells after splenectomy in immune thrombocytopenia patients

Distribution of peripheral blood cells after splenectomy in immune thrombocytopenia patients

Risk-based and individualised management of bleeding and thrombotic events in adults with primary immune thrombocytopenia (ITP).

Although bleeding is one of the main symptoms of primary immune thrombocytopenia (ITP), risk factors for bleeding have yet to be fully established. Low platelet count (PC; <20-30 × 109 /L) is generally indicative of increased risk of bleeding. However, PC and bleeding events cannot be fully correlated; many other patient- and disease-related factors are thought to contribute to increased bleeding risk. Furthermore, even though ITP patients have thrombocytopenia and are at increased risk of bleeding, ITP also carries higher risk of thrombotic events. Factors like older age and certain ITP treatments are associated with increased thrombotic risk. Women's health in ITP requires particular attention concerning haemorrhagic and thrombotic complications. Management of bleeding/thrombotic risk, and eventually antithrombotic therapies in ITP patients, should be based on individual risk profiles, using a tailored, patient-centric approach. Currently, evidence-based recommendations and validated tools are lacking to support decision-making and help clinicians weigh risk of bleeding against thrombosis. Moreover, evidence is lacking about optimal PC for achieving haemostasis in invasive procedures settings. Further research is needed to fully define risk factors for each event, enabling development of comprehensive risk stratification approaches. This review discusses risk-based and individualised management of bleeding and thrombosis risk in adults with primary ITP.

Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. For activation, 2.5 μM CaCl2 , 5 μM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

Regulatory role of ceRNA network in B lymphocytes of patients with immune thrombocytopenia

ObjectiveHigh-throughput sequencing was used to screen expressing differences of miRNA, lncRNA, and mRNA in CD19+ B peripheral blood samples of newly diagnosed immune thrombocytopenia (ITP) patients and healthy controls. The study aimed to explore the regulatory role of ceRNA network in the pathogenesis of dysfunctional CD19 + B lymphocytes of ITP patients.MethodsCD19+ B lymphocytes were isolated from peripheral blood samples of ITP patients and their healthy counterparts. High-throughput sequencing was used to screen for the expression of miRNA, lncRNA, and mRNA of ITP patients and healthy controls, which were analysed by the ceRNA network. Moreover, qPCR was used to verify the differential expression of miRNA, lncRNA, and mRNA in ITP patients and healthy controls. The correlation between differentially expressed miRNA, lncRNA, mRNA, and B lymphocyte subsets was also analysed.ResultsThe CD19+ B lymphocytes of 4 newly diagnosed ITP patients and 4 healthy controls were sequenced and analysed. There were 65 differentially expressed lncRNA and 149 mRNA forming a ceRNA network showed that 12 lncRNA and 136 differentially expressed mRNA were closely associated. Similarly, miR-144-3p, miR-374c-3p, and miR-451a were highly expressed in ITP patients, as confirmed by qPCR, which was consistent with the high-throughput sequence results. LOC102724852 and CCL20 were highly expressed in ITP patients, while LOC105378901, LOC112268311, ALAS2, and TBC1D3F were not as compared to healthy controls, which was consistent with the high-throughput sequence results. In addition, the expression of miR-374c-3p, LOC112268311, LOC105378901, and CXCL3 were correlated with the percentage of B lymphocyte subsets.ConclusionsThe ceRNA network of miRNA, lncRNA, and mRNA in peripheral CD19 + B lymphocytes plays an essential role in the pathogenesis of ITP.

Quercetin, a key active ingredient of Jianpi Zishen Xiehuo Formula, suppresses M1 macrophage polarization and platelet phagocytosis by inhibiting STAT3 activation based on network pharmacology.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease, and abnormal M1 macrophage polarization participates in the pathogenesis of ITP. Jianpi Zishen Xiehuo (JZX) Formula has a good therapeutic effect on ITP. However, its key active ingredients and molecular mechanisms remain unclear. In this study, we explored the key active ingredients and potential targets of JZX in treating ITP using network pharmacology combined with in vitro experimental verification. A total of 157 active ingredients of JZX were identified from public databases, and quercetin was the most important one. One hundred sixty-five intersection targets of active ingredients in JZX, ITP, and macrophage polarization were obtained by Venn diagram. The top three potential targets were signal transducer and activator of transcription 3 (STAT3), protein kinase B (PKB/AKT) 1, and c-JUN through protein-protein interaction analysis. Molecular docking showed that quercetin had strong binding affinities with them all. In vitro experiment, CD16+ monocytes increased in ITP patients compared with healthy controls, which indicated a M1/M2 polarization imbalance in ITP. The expression levels of M1 polarization markers, CD86, CD80, and inducible nitric oxide synthase (iNOS), M1 polarization-associated cytokines, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), and antibody-opsonized platelet phagocytosis significantly increased in THP-1 macrophages stimulated with lipopolysaccharide (LPS). Quercetin markedly inhibited the expressions of M1 markers, decreased the levels of TNF-α and IL-6, and down-regulated the phosphorylated STAT3 (p-STAT3) protein, which confirmed the prediction by network pharmacology and molecular docking. Importantly, quercetin significantly reduced the phagocytosis of antibody opsonised platelet. In conclusion, quercetin suppressed platelet phagocytosis in M1 macrophages via its anti-inflammatory effects and may serve as a potential drug forthe treatment of ITP. Quercetin could be a key ingredient for JZX against ITP.