Biomarker for evaluating prognosis condition of liver injury patient subjected to plasma exchange treatment and application of biomarker

Abstract

The invention relates to the technical field of bioengineering, in particular to a biomarker for evaluating the prognosis condition of a liver injury patient subjected to plasma exchange treatment and application of the biomarker. The biomarker comprises circulating mitochondrial DNA (deoxyribonucleic acid). Circulating mitochondrial DNA is used as a biomarker for evaluating the prognosis condition of the liver injury patient subjected to plasma exchange treatment, so that the prognosis early warning function of the liver injury patient subjected to plasma exchange can be realized, the survival rate of the patient is remarkably improved, and the technical blank of quickly and specifically monitoring the curative effect of plasma exchange is filled; the leap-forward progress of plasma exchange curative effect evaluation from empirical lag judgment to accurate real-time regulation is realized, and the method has remarkable clinical value and commercialization prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a biomarker for evaluating the prognosis of patients with liver damage after plasma exchange treatment and its application. Background Art

[0002] Plasma exchange, as an important extracorporeal blood purification technique, is widely used in the treatment of liver failure, autoimmune diseases, and toxic diseases. A standardized clinical application system has been established for the treatment of critical illnesses such as liver failure, but the technical principles, efficacy evaluation, and potential risks still require further exploration. Plasma exchange, as an important supportive treatment for liver failure, improves patient prognosis by clearing toxic substances and inflammatory factors from the body. However, its efficacy evaluation still relies on traditional liver function indicators and lacks precise molecular biological markers.

[0003] Currently, clinical evaluation of the efficacy of plasma exchange primarily relies on traditional biochemical markers (such as liver function indicators ALT, AST, and bilirubin) or clinical symptom observation (such as consciousness and bleeding tendency). Liver and kidney function indicators typically do not show significant changes until 24-48 hours after treatment, making real-time dynamic monitoring impossible during treatment. This results in a time lag and prevents timely adjustments to treatment plans (such as exchange volume and frequency). Traditional indicators reflect the cumulative effects of organ damage and are unable to specifically distinguish the efficiency of plasma exchange in clearing pathogens.

[0004] Recent studies have attempted to introduce inflammatory factors (such as IL-6 and TNF-α) as monitoring indicators, but they have the disadvantages of high detection cost, insufficient sensitivity and weak mechanism correlation.

[0005] Currently, there is no marker that can simultaneously meet the requirements of fast dynamic response, high specificity, and low-cost scalability. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a biomarker for evaluating the prognosis of patients with liver damage after plasma exchange treatment and its application.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a biomarker for evaluating the prognosis of patients with liver injury after plasma exchange treatment, wherein the biomarker comprises circulating mitochondrial DNA.

[0008] In a second aspect, the present invention provides a method for evaluating the prognosis of a patient with liver injury after plasma exchange treatment, the method comprising the following steps:

[0009] S1. Obtain blood samples before and after treatment from patients with liver injury receiving plasma exchange therapy.

[0010] S2, extracting plasma from the blood sample obtained in S1 and isolating DNA;

[0011] S3, quantitatively detecting the concentration of mitochondrial DNA in the DNA;

[0012] S4. Compare the changes in mitochondrial DNA concentration before and after treatment to evaluate the effect of plasma exchange therapy on the prognosis of patients with liver injury;

[0013] Among them, the significant reduction in mitochondrial DNA concentration in the blood of patients with liver injury after plasma exchange treatment indicates that the treatment is effective and the prognosis of patients with liver injury is improved.

[0014] Preferably, in step S3, the quantitative detection of the mitochondrial DNA concentration in the DNA adopts real-time PCR technology, using specific primers to amplify the D-LOOP loop of human mitochondrial DNA.

[0015] Preferably, the method further comprises jointly analyzing the mitochondrial DNA concentration and liver function indicators.

[0016] Preferably, the liver and kidney indicators include aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, γ-glutamyl transpeptidase, and cholinesterase.

[0017] In a third aspect, the present invention provides a kit for evaluating the prognosis of patients with liver damage after plasma exchange treatment, wherein the product comprises a reagent for detecting the biomarker according to claim 1.

[0018] Preferably, the reagents include reagents for isolating DNA from plasma, a fluorometer and detection reagents for quantifying mitochondrial DNA concentration, mitochondrial DNA-specific primers for real-time PCR amplification, and a reaction mixture.

[0019] In the above technical solution, the present invention innovatively combines the dynamic monitoring of mitochondrial DNA (mtDNA) concentration in plasma with the evaluation of plasma exchange treatment effects, establishes a correlation model between mtDNA quantitative detection and plasma exchange prognosis evaluation, and uses circulating mitochondrial DNA as a biomarker to evaluate the prognosis of patients with liver damage after plasma exchange treatment. It can not only realize the prognosis warning function for patients with liver damage who have undergone plasma exchange, significantly improve the survival rate of patients with liver damage, but also fill the technical gap in rapid and specific monitoring of the efficacy of plasma exchange, and realize a leap forward in the evaluation of the efficacy of plasma exchange from "empirical lagging judgment" to "precise real-time regulation", and has significant clinical value and commercial prospects.

[0020] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 The difference in plasma mtDNA concentration in patients with liver injury before and after plasma exchange treatment;

[0023] Figure 2 The difference in the levels of liver function indicators such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and total bilirubin (TBIL) before and after plasma exchange treatment in patients with liver injury;

[0024] Figure 3 The difference in the levels of liver function indicators direct bilirubin (DBIL), γ-glutamyl transpeptidase (GGT), and cholinesterase (CHE) in patients with liver injury before and after plasma exchange treatment;

[0025] Figure 4 Correlation between mtDNA level and ΔmtDNA in fresh frozen plasma (FFP);

[0026] Figure 5 The ROC curve of the prognostic diagnostic results of mtDNA detection alone in patients with liver injury treated with plasma exchange is shown;

[0027] Figure 6 The ROC curve of the prognostic diagnostic results of AST, ALT combined with mtDNA detection in patients with liver injury treated with plasma exchange was presented;

[0028] Figure 7 The ROC curve of the prognostic diagnostic results of TBIL, DBIL combined with mtDNA detection in patients with liver injury treated with plasma exchange was presented;

[0029] Figure 8 This is the ROC curve of the prognostic diagnostic results of GGT, CHE combined with mtDNA detection in patients with liver injury treated with plasma exchange. DETAILED DESCRIPTION

[0030] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0031] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0032] In a first aspect, the present invention provides a biomarker for evaluating the prognosis of a patient with liver injury treated with plasma exchange, wherein the biomarker comprises circulating mitochondrial DNA.

[0033] In a second aspect, the present invention provides a method for evaluating the prognosis of a patient with liver injury after plasma exchange treatment, the method comprising the following steps:

[0034] S1. Obtain blood samples before and after treatment from patients with liver injury receiving plasma exchange therapy.

[0035] S2, extracting plasma from the blood sample obtained in S1 and isolating DNA;

[0036] S3, quantitatively detecting the concentration of mitochondrial DNA in the DNA;

[0037] S4. Compare the changes in mitochondrial DNA concentration before and after treatment to evaluate the effect of plasma exchange therapy on the prognosis of patients with liver injury;

[0038] Among them, the significant reduction in mitochondrial DNA concentration in the blood of patients with liver injury after plasma exchange treatment indicates that the treatment is effective and the prognosis of patients with liver injury is improved.

[0039] In a preferred embodiment of the present invention, in step S3, the quantitative detection of the mitochondrial DNA concentration in the DNA adopts real-time PCR technology, using specific primers to amplify the D-LOOP loop of human mitochondrial DNA.

[0040] In a preferred embodiment of the present invention, the method further comprises jointly analyzing the mitochondrial DNA concentration and the liver and kidney function indicators.

[0041] In a preferred embodiment of the present invention, the liver and kidney indicators include aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, γ-glutamyl transpeptidase, and cholinesterase.

[0042] In a third aspect, the present invention provides a kit for evaluating the prognosis of patients with liver damage after plasma exchange treatment, wherein the product comprises a reagent for detecting the biomarker according to claim 1.

[0043] In a preferred embodiment of the present invention, the reagents include reagents for isolating DNA from plasma, a fluorometer and detection reagents for quantifying mitochondrial DNA concentration, mitochondrial DNA-specific primers for real-time PCR amplification, and a reaction mixture.

[0044] The present invention innovatively combines the dynamic monitoring of mitochondrial DNA (mtDNA) concentration in plasma with the evaluation of the therapeutic effect of plasma exchange, establishes a correlation model between mtDNA quantitative detection and plasma exchange prognosis evaluation, and uses circulating mitochondrial DNA as a biomarker to evaluate the prognosis of patients with liver damage after plasma exchange treatment. It can not only realize the prognosis warning function for patients with liver damage who undergo plasma exchange and significantly improve the survival rate of patients with liver damage, but also fill the technical gap in the rapid and specific monitoring of the efficacy of plasma exchange, and realize a leap-forward progress in the evaluation of the efficacy of plasma exchange from "empirical lagging judgment" to "precise real-time regulation", and has significant clinical value and commercial prospects.

[0045] The present invention will be described in detail below by way of examples. In the following examples, the drugs and pharmaceuticals are all conventional commercial products.

[0046] Method Description:

[0047] 1. Study population: This study included 23 patients who received plasma exchange (PE) treatment from January 2023 to December 2024. All patients were eligible for plasma exchange indications. Laboratory indicators were collected before and after plasma exchange treatment. However, during the study, 3 patients were excluded due to the following reasons:

[0048] (1) Two patients did not provide complete liver function data after plasma exchange treatment and were not included in the final analysis;

[0049] (2) One patient was discharged from the hospital due to clinical improvement after receiving one treatment and did not complete follow-up treatment and data collection, so he was not included in the analysis;

[0050] Finally, 20 patients completed the entire study process and were included in the data analysis.

[0051] 2. Inclusion and Exclusion Criteria

[0052] (1) Inclusion criteria: age over 18 years; all causes of plasma exchange indications, including myasthenia gravis, liver failure, renal failure, nervous system diseases, acute pancreatitis, and blood system diseases, are eligible for inclusion.

[0053] (2) Exclusion criteria: withdrawal of consent, presence of serious comorbidities (such as heart disease), recent treatment that affects mtDNA (such as chemotherapy), and inability to provide the data samples required for the study.

[0054] 3. Clinical detection indicators

[0055] Peripheral blood was collected before and 24 hours after treatment to test liver function indicators (such as AST, ALT, total bilirubin, direct bilirubin, GGT, CHE)

[0056] 4. Plasma mtDNA detection method

[0057] The collected patient blood samples were centrifuged at 1600g for 10 min, and after removing the plasma supernatant, they were centrifuged again at 16,000g for 10 min to remove apoptotic bodies;

[0058] DNA was isolated from 200 μL of plasma using the QIAamp DNAMini Kit (Qiagen) and eluted with 50 μL of ultrapure water, and DNA samples were stored at −20°C until analysis;

[0059] The total DNA concentration of cfDNA was quantified using the Qubit Fluorometer (Invitrogen) and the Qubit dsDNAHS Assay Kit;

[0060] The subcellular origin of cfDNA was quantified by real-time PCR using the SsoAdvanced SYBR Green Supermix (Bio-Rad) and the QuantStudio 7Flex system (Applied Biosystems). Primers were used to amplify the human β-globin gene and the D-Loop loop, respectively, for quantification of nc-cfDNA (ncDNA) and mt-cfDNA, respectively, using the following thermal cycling conditions: 98°C for 3 minutes; 98°C for 15 seconds; 47°C for 30 seconds; and 60°C for 30 seconds.

[0061] 5. Statistical methods

[0062] SPSS 27.0 statistical version was used to perform descriptive statistical analysis on the basic demographic characteristics of the sample, and the results were presented as frequencies and percentages (%) of each group.

[0063] Microscientific software was used to draw the graphical display of the T-test results, and the statistical significance level was set at P < 0.05 to determine whether the differences between the groups were significant.

[0064] The receiver operating characteristic (ROC) curve was used for predictive efficiency analysis. P < 0.05 indicated that the difference was statistically significant. It is generally believed that the area under the ROC curve between 0.50-0.70 indicates low diagnostic accuracy, between 0.70-0.90 indicates moderate diagnostic accuracy, and above 0.90 indicates high diagnostic accuracy.

[0065] Example 1

[0066] This example is used to illustrate the overall condition of the patients involved in the present invention.

[0067] Statistical analysis was performed on the patients included in the study, including percentages and frequencies. The overall situation of the study sample was analyzed by patient gender, age, diseases and complications. In this description, 20 study samples were included, including 6 females (30%) and 14 males (70%). According to age group, 25-40 years old accounted for 15%, 40-55 years old accounted for 30%, 55-70 years old accounted for 35%, and 70-85 years old accounted for 20%. In addition, the distribution of disease types showed that the prevalence of acute liver failure was as high as 50%, and complications showed that the probability of hepatitis B cirrhosis and hypoproteinemia was high, accounting for 30% and 25% respectively. It was highlighted that the liver damage was more prominent, which provided valuable background information for subsequent analysis. The specific situation is shown in Table 1.

[0068] Table 1

[0069]

[0070]

[0071] Example 2

[0072] This example is used to illustrate the difference in mitochondrial DNA levels in the plasma of patients treated with PE before and after treatment.

[0073] Depend on Figure 1 The results showed that the concentration of mtDNA in the plasma of patients after PE treatment showed a gradual downward trend during the treatment process. The mtDNA data of patients' plasma before and after treatment were included in the T test analysis, and the treatment effect was very significant (P<0.05), and the difference was statistically significant.

[0074] Example 3

[0075] This example is used to illustrate the difference in liver function levels before and after PE treatment in patients.

[0076] Depend on Figure 2-Figure 3 It can be seen that the liver function indicators of patients treated with PE before and after treatment, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), direct bilirubin (DBIL), γ-glutamyl transpeptidase (GGT) and cholinesterase (CHE), were analyzed by T test. It can be seen that the effect of plasma treatment on liver injury was significant (P<0.05), and the difference was statistically significant.

[0077] Example 4

[0078] This example is used to illustrate the results of prognostic diagnosis of patients with liver damage treated with plasma exchange using mtDNA alone. Figure 5 As shown in the figure, the results of combined detection of liver function indicators and mtDNA for prognostic diagnosis of patients with liver injury treated with plasma exchange are as follows: Figure 6-Figure 8 shown.

[0079] Depend on Figure 5-Figure 8 It can be seen that the ROC curve analysis results of the present invention show that the AUC of the combined mtDNA and liver function indicators is greater than the AUC of the individual mtDNA and liver function indicators, and the difference is statistically significant (P<0.05).

[0080] The area under the ROC curve (AUC) indicates the accuracy of the results. The AUC value ranges from 0 to 1. The larger the value, the higher the accuracy of the model results. It is generally believed that an area under the ROC curve between 0.50 and 0.70 indicates low diagnostic accuracy, between 0.70 and 0.90 indicates medium diagnostic accuracy, and above 0.90 indicates high diagnostic accuracy.

[0081] The AUC values ​​of the ROC curve of the treatment prognosis response prediction model based on mtDNA and its combination with liver function indicators in the present invention were 0.858, 0.760, 0.793, 0.769, 0.793, 0.81, and 0.777, respectively, while the AUC values ​​of the combined mtDNA and liver function indicators were 0.901, 0.950, 0.893, 0.860, 0.917, and 0.901 (all P < 0.05), as shown in Table 2. This shows that the model results are accurate and can better reflect the high AUC value of the combined mtDNA and liver function indicators, which may make it a reliable biomarker combination for predicting the prognosis effect of plasma exchange treatment.

[0082] Table 2

[0083]

[0084]

[0085] Among them, a is based on non-parametric assumptions; b is the original hypothesis: true area = 0.5.

[0086] Example 5

[0087] This example is used to illustrate the relationship between the mtDNA level in fresh frozen plasma (FFP) and the efficacy of plasma exchange.

[0088] Contrary to the initial assumption that FFP could replace plasma from patients with high mtDNA concentrations, total FFP was collected from 11 patients whose liver function improved during plasma exchange.

[0089] Depend on Figure 4The results show that there is no significant correlation between the change in mtDNA (ΔmtDNA) before and after PE treatment and the mtDNA level in FFP. At the same time, as shown in Table 3, there is no significant correlation between the mtDNA concentration in FFP and liver function treatment. This indicates that plasma exchange does not directly reduce the mtDNA level in the patient's body, but reduces the mtDNA level after treatment by improving the patient's disease status and reducing tissue damage.

[0090] A normal human body has a certain amount of mtDNA, and fresh frozen plasma also contains a small amount of white blood cells. The higher level of mtDNA detected in FFP should come from the rupture of white blood cells during the thawing process, releasing a certain amount of mtDNA. It is speculated that this is because FFP has a certain concentration of mtDNA.

[0091] Table 3

[0092]

[0093] In summary, the present invention demonstrates for the first time through clinical and experimental evidence that plasma exchange can effectively reduce circulating mtDNA, and its dynamic changes are closely related to liver function recovery and prognosis. Although mtDNA participates in inflammatory responses as a DAMP, its core role in PE treatment is as a marker of efficacy rather than an active pathogenic factor: on the one hand, the decrease in mtDNA levels after treatment occurs synchronously with the improvement of liver function, suggesting that changes in its concentration reflect the reduction of the inflammatory load in the body; on the other hand, mtDNA from blood donors does not show stronger pro-inflammatory activity, and the infusion of exogenous mtDNA does not aggravate or alleviate tissue damage, indicating that the therapeutic benefits of PE mainly come from the clearance of the patient's own pathogenic substances, rather than blocking the inflammatory cascade by changing the characteristics of the donor's mtDNA. This provides a new molecular target for the clinical and precise evaluation of the efficacy of PE - by monitoring the dynamic changes of mtDNA, the regulatory effect of treatment on the inflammatory microenvironment in the body can be judged in real time.

[0094] The present invention innovatively combines the dynamic monitoring of mitochondrial DNA (mtDNA) concentration in plasma with the evaluation of the therapeutic effect of plasma exchange, establishes a correlation model between mtDNA quantitative detection and plasma exchange prognosis evaluation, and uses circulating mitochondrial DNA as a biomarker to evaluate the prognosis of patients with liver damage after plasma exchange treatment. It can not only realize the prognosis warning function for patients with liver damage who undergo plasma exchange and significantly improve the survival rate of patients with liver damage, but also fill the technical gap in the rapid and specific monitoring of the efficacy of plasma exchange, and realize a leap-forward progress in the evaluation of the efficacy of plasma exchange from "empirical lagging judgment" to "precise real-time regulation", and has significant clinical value and commercial prospects.

[0095] In the prior art, when treating liver injury-related diseases, there is no clear conclusion or gold standard for the optimal dosage and number of plasma exchange treatments. Most of the results are based on years of clinical experience, the patient's specific condition, and the patient's response. The present invention uses mtDNA as a non-invasive and immediate biomarker, which can detect changes in its levels in plasma to reflect the cell damage of patients with liver injury in real time, and then judge the disease progression and the treatment effect of PE. Especially in complex clinical situations, by combining real-time mtDNA detection results, doctors can more accurately monitor the prognosis of patients with liver injury treated with PE, avoid overtreatment or undertreatment, and thus improve the accuracy of treatment.

[0096] In summary, plasma exchange, as an effective treatment, has made certain progress in research both domestically and internationally. The present invention uses mtDNA as a biomarker to assess the prognosis of patients with liver damage after plasma exchange treatment, providing a portable, accurate, and non-invasive means to evaluate the effectiveness of plasma exchange treatment. The combination of plasma exchange treatment and mtDNA not only helps to provide more personalized treatment plans, but also enables real-time monitoring of treatment effects, improves the speed of patient recovery and the safety of treatment, enables more accurate disease management, and promotes the development of personalized and precise medical treatment.

[0097] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0098] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0099] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A biomarker for evaluating the prognosis of patients with liver injury after plasma exchange treatment, characterized in that: The biomarkers include circulating mitochondrial DNA.

2. A method for evaluating the prognosis of a patient with liver damage after plasma exchange treatment, characterized in that: The method comprises the following steps: S1. Obtain blood samples before and after treatment from patients with liver injury receiving plasma exchange therapy; S2, extracting plasma from the blood sample obtained in S1 and isolating DNA; S3, quantitatively detecting the concentration of mitochondrial DNA in the DNA; S4. Compare the changes in mitochondrial DNA concentration before and after treatment to evaluate the effect of plasma exchange therapy on patient prognosis; Among them, the significant reduction in mitochondrial DNA concentration in the blood of patients with liver injury after plasma exchange treatment indicates that the treatment is effective and the prognosis of patients with liver injury is improved.

3. The method according to claim 2, characterized in that In step S3, the quantitative detection of the mitochondrial DNA concentration in the DNA adopts real-time PCR technology, and uses specific primers to amplify the D-LOOP loop of human mitochondrial DNA.

4. The method according to claim 2 or 3, characterized in that The method further comprises analyzing the mitochondrial DNA concentration in combination with liver function indicators.

5. The method according to claim 4, characterized in that The liver and kidney indicators include aspartate aminotransferase, alanine aminotransferase, total bilirubin, direct bilirubin, γ-glutamyl transpeptidase, and cholinesterase.

6. A kit for evaluating the prognosis of patients with liver damage after plasma exchange treatment, characterized in that: The product comprises a reagent for detecting the biomarker according to claim 1.

7. The kit according to claim 6, characterized in that The reagents include reagents for separating DNA from plasma, a fluorometer and detection reagents for quantitatively determining the concentration of mitochondrial DNA, and mitochondrial DNA-specific primers and a reaction mixture for real-time PCR amplification.

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