Effects of thrombopoietin pre-treatment on peri-liver transplantation thrombocytopenia in a mouse model of cirrhosis with hypersplenism

Zi-Rong Liu,Ya-Min Zhang,Zi-Lin Cui,Wen Tong

Abstract BACKGROUND During cirrhosis,the liver is impaired and unable to synthesize and clear thrombopoietin properly.At the same time,the spleen assumes the function of hemofiltration and storage due to liver dysfunction,resulting in hypersplenism and excessive removal of platelets in the spleen,further reducing platelet count.When liver function is decompensated in cirrhotic patients,the decrease of thrombopoietin (TPO) synthesis is the main reason for the decrease of new platelet production.This change of TPO leads to thrombocytopenia and bleeding tendency in cirrhotic patients with hypersplenism.AIM To investigate the clinical efficacy of recombinant human TPO (rhTPO) in the treatment of perioperative thrombocytopenia during liver transplantation in cirrhotic mice with hypersplenism.METHODS C57BL/6J mice and TPO receptor-deficient mice were used to establish models of cirrhosis with hypersplenism.Subsequently,these mice underwent orthotopic liver transplantation (OLT).The mice in the experimental group were given rhTPO treatment for 3 consecutive days before surgery and 5 consecutive days after surgery,while the mice in the control group received the same dose of saline at the same frequency.Differences in liver function and platelet counts were determined between the experimental and control groups.Enzyme-linked immunosorbent assay was used to assess the expression of TPO and TPO receptor(c-Mpl) in the blood.RESULTS Preoperative administration of rhTPO significantly improved peri-OLT thrombocytopenia in mice with cirrhosis and hypersplenism.Blocking the expression of TPO receptors exacerbated peri-OLT thrombocytopenia.The concentration of TPO decreased while the concentration of c-Mpl increased in compensation in the mouse model of cirrhosis with hypersplenism.TPO pre-treatment significantly increased the postoperative TPO concentration in mice,which in turn led to a decrease in the c-Mpl concentration.TPO pre-treatment also significantly enhanced the Janus kinase(Jak)/signal transducers and activators of transcription pathway protein expressions in bone marrow stem cells of the C57BL/6J mice.Moreover,the administration of TPO,both before and after surgery,regulated the levels of biochemical indicators,such as alanine aminotransferase,alkaline phosphatase,and aspartate aminotransferase in the C57BL/6J mice.CONCLUSION Pre-treatment with TPO not only exhibited therapeutic effects on perioperative thrombocytopenia in the mice with cirrhosis and hypersplenism,who underwent liver transplantation but also significantly enhanced the perioperative liver function.

Key Words: Thrombopoietin pre-treatment;Cirrhosis;Liver transplantation;Perioperative period;Platelet

INTRODUCTION

Thrombocytopenia in cirrhosis is a rare condition with various causes including hypersplenism,reduced levels of thrombopoietin (TPO),presence of anti-platelet autoantibodies,suppression of bone marrow cells by hepatitis viruses,adverse reactions to excessive alcohol intake,liver dysfunction,and vitamin B12 and folic acid deficiencies.Of these,hypersplenism is the most common cause.However,for patients with cirrhosis in the decompensated stage,a decrease in TPO production by hepatocytes is the main cause for the reduced production of new platelets[1-3].Most patients with cirrhosis and hypersplenism,who are candidates for orthotopic liver transplantation (OLT),are in the decompensated stage,with comorbidities,such as hepatitis or a long history of excessive alcohol consumption.As a result,these patients exhibit more pronounced hypersplenism and have a slower postoperative recovery compared to those with cirrhosis and hypersplenism alone.A study of patients with liver failure,before and after liver transplantation,found that all patients manifested prominent thrombocytopenia before transplantation,with serum levels so low that TPO was undetectable.In contrast,the serum TPO levels increased significantly 2 d after transplantation and reached their peaks at 4-6 d.At the same time,platelet counts began to rise and peaked at 14 d after transplantation.Furthermore,other cytokines affecting platelet production did not change significantly before and after transplantation[4,5].These findings suggest that,in patients with liver failure,TPO plays a critical role in thrombocytopenia and in the restoration of platelet count after liver transplantation.Gollompet al[6] found similar serum TPO levels before and after liver transplantation,as well as significantly lower TPO mRNA expressions in the liver tissues of patients with cirrhosis.Thrombocytopenia in patients with liver failure is challenging.Current research to improve the postoperative platelet counts of these patients and,thus,reduce the risk of bleeding and promote rapid recovery from hypersplenism is necessary.

MATERIALS AND METHODS

Study of the treatment efficacy of TPO for perioperative thrombocytopenia in mice undergoing liver transplantation for cirrhosis with hypersplenism

A mouse model of liver cirrhosis with hypersplenism was established by weighing the mice and injecting them intraperitoneally with carbon tetrachloride (CCl4,1 mL/kg body weight) three times a week for 6 wk.At the end of the 6 wk,blood was collected from the orbital vein to determine the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT),and the liver tissues were stained with hematoxylin and eosin (HE) to determine whether the model was successfully developed.

Groups:C57BL/6J mice were used to establish a model of cirrhosis with hypersplenism.These mice subsequently underwent OLT.The experimental group received recombinant human TPO (rhTPO) treatment for 3 consecutive days before surgery and 5 consecutive days after surgery,while the control group received saline instead at the same dose and frequency.

Sample collection:Blood samples were collected 1 d before and 5 d after surgery.Liver tissue samples and bone marrow mesenchymal stem cells were collected from euthanized experimental and control mice 5 d after surgery.

Assessed indicators:Changes in liver function [ALT,AST,and alkaline phosphatase (ALP) levels] were assessed by biochemical assays at different time points.Changes in the level of leukocytes,erythrocytes,platelets,etc.were detected by routine blood tests at different time points.Pathological liver changes were observed in the tissue sections at different time points.Serum TPO and c-Mpl levels were determined using enzyme-linked immunosorbent assay (ELISA).The expression of Jak/signal transducers and activators of transcription (STAT),in extracted mouse bone marrow mesenchymal stem cells,was detected with Western blotting.

Study on whether blocking the expression of TPO receptor can exacerbate perioperative thrombocytopenia

Groups:Mice with a silenced TPO receptor gene and C57BL/6J mice were used to establish the model of cirrhosis with hypersplenism.These mice then all underwent OLT.The experimental group received rhTPO treatment for 3 consecutive days before surgery and 5 consecutive days after surgery,while the control group received saline instead at the same dose and frequency.

Sample collection:Same as described above.

Assessed indicators:Same as described above.

Statistical analysis

SPSS,version 22.0 (IBM Corp.,Armonk,NY,United States) was used for analysis.Normally distributed quantitative data were expressed as mean ± SD,and at-test was used for comparison between the groups.The data with skewed distributions were expressed as M (range),and a Mann-WhitneyUtest was used for comparison between the groups.Count data were expressed as absolute numbers or percentages,and aχ²test or a Fisher’s exact test was used for comparisons between the groups.Data with repeated measurements were analyzed using repeated-measures analysis of variance(ANOVA).P＜ 0.05 was considered statistically significant.

RESULTS

Study of the treatment efficacy of TPO for perioperative thrombocytopenia in mice undergoing liver transplantation for cirrhosis with hypersplenism

C57BL/6J mice were treated with CCl4 to establish a model of cirrhosis with hypersplenism.Subsequently,these mice underwent OLT.In the experimental group,rhTPO was administrated for 3 consecutive days before and 5 consecutive days after surgery,while in the control group,an equal amount of saline was administrated at the same frequency instead.Routine blood tests and liver function assessments of the experimental and control mice were performed 1 d before and 5 d after surgery to monitor the changes.The results showed that TPO pre-treatment significantly ameliorated erythropenia and thrombocytopenia during the perioperative period of liver transplantation in the experimental C57BL/6 mice compared to the control mice (Figure 1A and C).However,the leukocyte count was not affected by TPO pretreatment (Figure 1B).Importantly,TPO pre-treatment significantly improved the perioperative liver function of these mice (Figure 1D-F).In contrast,TPO receptor-deficient mice responded poorly to TPO pre-treatment compared to C57BL/6J mice.As such,TPO pre-treatment failed to elevate the perioperative platelet counts in TPO receptor-deficient mice (Figure 2A and B).

Figure 1 Comparison of routine blood and biochemistry results between the experimental and control groups during the perioperative period of liver transplantation. The experimental group was treated with thrombopoietin,while the control group was treated with an equal amount of saline.aP＜ 0.05;bP ＜ 0.01.ALT: Alanine aminotransferase;ALP: Alkaline phosphatase;AST: Aspartate aminotransferase.

Figure 2 Evaluation of the responsiveness to thrombopoietin pre-treatment and Differences in thrombopoietin and c-Mpl levels. A and B:Evaluation of the responsiveness to thrombopoietin pre-treatment in thrombopoietin receptor-deficient mice and C57B/6J mice during the perioperative period of liver transplantation;C and D: Differences in thrombopoietin and c-Mpl levels between the experimental and control groups during the perioperative period of liver transplantation determined by enzyme-linked immunosorbent assay.The experimental group was treated with thrombopoietin,while the control group was treated with an equal amount of saline.aP ＜ 0.05;bP ＜ 0.01.TPO: Thrombopoietin.

ELISA to determine the differences in TPO and c-Mpl levels

ELISA was used to determine the differences in TPO and c-Mpl levels between the experimental and control groups,1 d before and 5 d after surgery.Compared to the control group,the results from the experimental group showed that the TPO treatment significantly increased serum TPO after liver transplantation,which in turn,led to a decrease in the c-Mpl level (Figure 2C and D).

HE staining and differential analysis of protein expression after liver transplantation

HE staining showed that the mice in the experimental group which received TPO pre-treatment had significantly reduced inflammatory responses in the liver tissues compared to those in the control group (Figure 3).Considering that the Jak/STAT pathway plays an important role in the differentiation and maturation of megakaryocytes and the promotion of platelet production,we examined the differential expression of the Jak/STAT pathway proteins between the TPO receptor-deficient mice and the C57BL/6J mice.TPO pre-treatment significantly enhanced the expression of the Jak/STAT pathway proteins in the bone marrow stem cells of C57BL/6J mice compared to those of the TPO receptor-deficient mice (Figure 4).

Figure 3 Comparison of the HE-stained liver tissues between the experimental and control groups after liver transplantation. The experimental group was treated with thrombopoietin,while the control group was treated with an equal amount of saline.The arrows indicate the area of inflammatory necrosis.

DISCUSSION

TPO is a glycoprotein with 332 amino acids produced mainly by hepatocytes and is an endogenous cytokine that stimulates the growth and differentiation of megakaryocytes.It has stimulative effects on megakaryopoiesis at all stages,including the proliferation of precursor cells and the development and maturation of polyploid megakaryocytes.Once TPO binds to its receptor,c-Mpl,it induces the homodimerization of c-Mpl,activating the family of JAK in signaling pathways,including Jak/STAT,P13K/Akt,Ras/MAPK,etc.,and the secretion of a series of signaling molecules to induce megakaryocyte differentiation and maturation and promote platelet production.The secretion of TPO is mainly influenced by the number of peripheral blood platelets,and TPO,in turn,acts to maintain a stable number of peripheral blood platelets[7-10].rhTPO is a purified full-length glycosylated TPO produced by Chinese hamster ovary cells that are modified using recombinant gene technology.It has the same amino acid sequence as TPO and is fully glycosylated with similar platelet-elevating pharmacological effects as endogenous TPO[11-13].Most patients with cirrhosis and hypersplenism who require OLT are in a decompensated stage of cirrhosis,with comorbidities,such as hepatitis or a long history of excessive alcohol consumption.As such,thrombocytopenia tends to be more severe,and postoperative recovery is usually slower in these patients than those with cirrhosis and hypersplenism alone.However,the therapeutic effects of rhTPO in these patients have not yet been elucidated.

Several studies have employed in-situ hybridization techniques to demonstrate that the liver is the primary organ expressing mRNA of TPO,despite the expression of TPO mRNA in other organs[14-16].It is now confirmed that the main site of TPO production in the body is the liver.Under physiological conditions,TPO is cleared from the blood by binding to its receptors on the platelet surface.As the platelet count increases,TPO clearance also increases,resulting in a decreasing blood TPO level.Conversely,blood TPO level increases when the platelet count decreases[17,18].There are two major contributors to thrombocytopenia in cirrhosis: Abnormal platelet distribution and a decreased production of hepatic TPO.Abnormal platelet distribution is related to the circuiting platelets being trapped in the spleen,and the degree of entrapment is positively associated with the size of the spleen.The decreased production of hepatic TPO is related to TPO production in organs,specifically 70% of TPO originates from the liver and only 30% originates from the kidneys and other organs.The indications for rhTPO during the per-OLT period in patients with cirrhosis and hypersplenism are as follows: (1) Patients with hypersplenism who have reduced circulating platelets before liver transplantation,leading to a high risk of intra-and postoperative bleeding.In this case,pre-and postoperative administration of rhTPO can appropriately raise the level of platelets to reduce the risk of surgical bleeding,with a long-lasting effect even after discontinuation as rhTPO has a long half-life;and (2) patients in the initial period following liver transplantation.During this critical time,the patient’s liver function remains impaired,resulting in reduced TPO production.Moreover,hypersplenism is still present in these patients since the condition ameliorates very slowly.Therefore,these patients also have a high risk of postoperative bleeding.The continuous administration of rhTPO after surgery can promote platelet production,while promoting the release of functional circulating platelets to reduce the liver burden,promote rapid restoration of liver function,and lay a good foundation for hepatic TPO production.The restoration of liver function can also reduce portal pressure,promote rapid remission of hypersplenism,and reduce platelet phagocytosis,thus forming a virtuous circle.

The present study found that preoperative administration of rhTPO significantly improved peri-liver transplantation thrombocytopenia in mice with cirrhosis and hypersplenism.Blocking the expression of TPO receptors exacerbated peri-OLT thrombocytopenia.The concentration of TPO decreased,while the concentration of c-Mpl increased in compensation,in the mouse model of cirrhosis with hypersplenism.TPO pre-treatment significantly increased the postoperative TPO concentration in mice,which in turn,led to a decrease in the c-Mpl concentration.TPO pre-treatment significantly enhanced the expressions of proteins involved in the Jak/STAT pathway in the bone marrow stem cells of C57BL/6J mice,which is consistent with the results from other studies.Additionally,two animal studies have shown that TPO can promote liver regeneration and ameliorate liver fibrosis by promoting platelet production[19,20].A clinical study has also shown that platelet transfusion can improve liver function in patients with chronic liver disease and cirrhosis.In the present study,we demonstrated that TPO can regulate the levels of the biochemical indicators,such as ALT,ALP,and AST in C57BL/6J mice,regardless of the timing of its administration (before or after surgery).Our results also validated that TPO can improve liver function in mice by enhancing the effects of platelets.However,there are still some limitations and deficiencies in this study.First,the study was limited to a mouse model,and the clinical effects of TPO on perioperative liver transplant patients need to be further explored in the future.In addition,the study found that TPO exerts pharmacological effects by activating the Jak/Stat3 pathway,and the specific molecular mechanisms in this pathway still need to be further demonstrated by basic experiments.

CONCLUSION

In conclusion,we found that preoperative prophylactic use of TPO has a therapeutic effect on perioperative thrombocytopenia in cirrhotic hyper splenic mice undergoing liver transplantation.In addition,TPO pretreatment can significantly improve the liver function of perioperative mice.TPO pretreatment also improved postoperative liver inflammation and reduced liver cell necrosis in mice.

ARTICLE HIGHLIGHTS

Research background

During cirrhosis,the liver undergoes significant impairment,leading to various complications,including thrombocytopenia and bleeding tendency.Thrombopoietin (TPO) is a hormone produced by the liver that plays a crucial role in regulating platelet production and clearance.However,in cirrhotic patients,the liver’s ability to synthesize and clear TPO is compromised.The impaired liver function in cirrhosis results in reduced TPO synthesis.TPO is primarily produced in the liver sinusoidal endothelial cells,and when the liver is damaged,the production of TPO is significantly decreased.This reduction in TPO levels leads to a decrease in the production of new platelets in the bone marrow,contributing to thrombocytopenia.

Research motivation

It is important to manage thrombocytopenia and bleeding tendency in cirrhotic patients.Treatment options may include platelet transfusions,medications that stimulate platelet production (such as TPO receptor agonists),and interventions to address the underlying liver dysfunction.Close monitoring and collaboration with a healthcare provider are crucial in managing these complications in cirrhotic patients.

Research objectives

To evaluate the clinical effectiveness of recombinant human TPO (rhTPO) in managing perioperative thrombocytopenia during liver transplantation in cirrhotic mice with hypersplenism.We aimed to assess whether rhTPO administration could effectively increase platelet count and reduce bleeding complications in this specific population.

Research methods

To achieve this objective,we conducted a controlled experiment using a cirrhotic mouse model with hypersplenism.The mice were divided into two groups: A treatment group receiving rhTPO and a control group receiving a placebo or standard care.We monitored the platelet counts of the mice before and after liver transplantation,as well as during the perioperative period.

Research results

The results of our study demonstrated that preoperative administration of rhTPO effectively improved perioperative thrombocytopenia in mice with cirrhosis and hypersplenism undergoing liver transplantation (OLT).This finding suggests that rhTPO may have potential clinical efficacy in managing thrombocytopenia in cirrhotic patients undergoing liver transplantation.Furthermore,we found that blocking the expression of TPO receptors exacerbated peri-OLT thrombocytopenia,indicating the importance of the TPO/c-Mpl pathway in platelet regulation during liver transplantation in cirrhotic mice with hypersplenism.In our study,we observed a decrease in TPO concentration in the mouse model of cirrhosis with hypersplenism,while the concentration of c-Mpl increased in compensation.However,pre-treatment with TPO significantly increased the postoperative TPO concentration in mice,leading to a decrease in the c-Mpl concentration.This suggests that TPO administration can regulate the TPO/c-Mpl pathway and potentially improve platelet production and function.Additionally,TPO pre-treatment significantly enhanced the protein expressions of the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway in bone marrow stem cells of the mice.The Jak/STAT pathway is involved in regulating various cellular processes,including cell proliferation and differentiation,and plays a role in platelet production.The enhancement of this pathway may contribute to the increased platelet production observed with TPO administration.Moreover,the administration of TPO,both before and after surgery,was found to regulate the levels of biochemical indicators,such as alanine aminotransferase (ALT),alkaline phosphatase (ALP),and aspartate aminotransferase (AST) in the mice.This suggests that TPO administration may have additional beneficial effects on liver function and overall liver health in cirrhotic mice undergoing liver transplantation.

Research conclusions

Pre-treatment with TPO not only exhibited therapeutic effects on perioperative thrombocytopenia in the mice with cirrhosis and hypersplenism,who underwent liver transplantation but also significantly enhanced the perioperative liver function.

Research perspectives

Overall,our study provides evidence supporting the clinical efficacy of rhTPO in managing perioperative thrombocytopenia during liver transplantation in cirrhotic mice with hypersplenism.These findings may have implications for the development of potential therapeutic strategies for managing thrombocytopenia in cirrhotic patients undergoing liver transplantation.

FOOTNOTES

Author contributions:Liu ZR,Cui ZL,and Tong W designed the research study;Liu ZR and Cui ZL performed the research;Liu ZR and Tong W contributed new reagents and analytic tools;Liu ZR and Cui ZL analyzed the data and wrote the manuscript;Zhang YM is responsible for reviewing the entire study;and all authors have read and approve the final manuscript.

Institutional animal care and use committee statement:All procedures involving animals were reviewed and approved by the Tianjin Tiancheng New Drug Evaluation Co.,Ltd (Approval No.2023041701).

Conflict-of-interest statement:All authors declare that there are no conflicts of interest.

Data sharing statement:The data of this study can be shared.

ARRIVE guidelines statement:The authors have read the ARRIVE Guidelines,and the manuscript was prepared and revised according to the ARRIVE Guidelines.

Open-Access:This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers.It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license,which permits others to distribute,remix,adapt,build upon this work non-commercially,and license their derivative works on different terms,provided the original work is properly cited and the use is non-commercial.See: https://creativecommons.org/Licenses/by-nc/4.0/

Country/Territory of origin:China

ORCID number:Zi-Rong Liu 0000-0002-1731-0035;Ya-Min Zhang 0000-0001-7886-2901;Wen Tong 0000-0002-2067-8121.

S-Editor:Chen YL

L-Editor:A

P-Editor:Chen YL