A hematological marker for diagnosing sarcopenia in patients with chronic obstructive pulmonary disease

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a hematological marker for diagnosing sarcopenia in patients with chronic obstructive pulmonary disease. The present invention applies the hematological marker resistin to the diagnosis of sarcopenia in patients with COPD for the first time. The expression level of the hematological marker resistin shows significant differences between COPD patients with sarcopenia and COPD patients with normal muscle function; the resistin expression level is significantly correlated with the muscle function of COPD patients; ROC curve analysis shows that it has high specificity and sensitivity as a detection variable. The present invention provides a new target for the diagnosis of sarcopenia in COPD, which has great clinical significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a hematological marker for diagnosing sarcopenia in patients with chronic obstructive pulmonary disease. Background Art

[0002] Sarcopenia is mainly manifested as progressive and widespread loss of muscle function. Specifically, it manifests as: decreased muscle mass, decreased muscle strength, and weakened muscle oxidative capacity. Chronic obstructive pulmonary disease (COPD) is a common preventable and treatable disease characterized by persistent respiratory symptoms and airflow limitation, usually due to exposure to toxic particles or gases. Sarcopenia is one of the important comorbidities in COPD patients and an independent risk factor for increased mortality. COPD patients with sarcopenia have significantly decreased exercise capacity, and insufficient exercise tolerance further aggravates muscle dysfunction. Sarcopenia is an independent risk factor for increased mortality in COPD patients.

[0003] At present, the diagnosis of sarcopenia mainly relies on the consensus on the diagnostic criteria for sarcopenia issued by the European Elderly Sarcopenia Working Group and the Asian Sarcopenia Working Group. Both recommend the combined measurement of skeletal muscle mass (by dual-energy X-ray or bioelectrical impedance analysis) and muscle function (grip strength and walking speed) to diagnose sarcopenia. The diagnostic process of sarcopenia requires specific equipment and professional training, which is not convenient in developing countries and primary care institutions. Therefore, serum biomarkers based on blood test results will help predict or identify sarcopenia in patients with COPD.

[0004] Resistin is a cysteine-rich, fat-derived peptide hormone encoded by the RETN gene in humans. In primates, resistin is secreted by immune cells and epithelial cells. Several studies have shown that resistin is an endogenous ligand for Toll-like receptor 4 and is involved in the activation of multiple inflammatory signaling pathways. A large number of studies have shown that resistin is a key inflammatory cytokine involved in many chronic diseases, such as atherosclerosis, tumors, and diabetic vascular disease. Compared with healthy controls, the average resistin level in the serum of COPD patients is 2 times higher. In addition, the forced expiratory volume in 1 second (FEV1) of COPD patients is significantly negatively correlated with resistin levels. However, to date, no studies other than the applicant of this patent have examined whether resistin can be used as a serological marker for sarcopenia in COPD patients. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a hematological marker for screening sarcopenia in COPD patients. The expression level of the hematological marker resistin shows significant differences between COPD patients with sarcopenia and COPD patients with normal muscle function; the expression level of resistin is significantly correlated with the muscle function of COPD patients; ROC curve analysis shows that it has high specificity and sensitivity as a detection variable.

[0006] In order to achieve the above object, the present invention provides the following technical solutions.

[0007] The present invention provides the use of a reagent for detecting the expression level of resistin in the preparation of an auxiliary diagnosis product for chronic obstructive pulmonary disease combined with sarcopenia.

[0008] Furthermore, the auxiliary diagnostic product for chronic obstructive pulmonary disease combined with sarcopenia uses resistin as a marker.

[0009] Furthermore, the expression level of resistin is positively correlated with the incidence of chronic obstructive pulmonary disease combined with sarcopenia.

[0010] Furthermore, the product detects the expression level of resistin in a sample by reverse transcription PCR, real-time quantitative PCR, in situ hybridization, Northern blotting, chip, high-throughput sequencing platform, immunohistochemical staining or enzyme-linked immunosorbent assay.

[0011] Furthermore, the product contains specific primers for amplifying the resistin gene, probes that hybridize with the nucleotide sequence of the resistin gene, or antibodies that specifically bind to resistin.

[0012] Furthermore, the product is a chip, a preparation or a kit.

[0013] Furthermore, the sample is serum.

[0014] Compared with the prior art, the present invention has the following beneficial effects.

[0015] 1) This invention is the first time that the hematological marker resistin is used in the diagnosis of patients with COPD sarcopenia, which is innovative in its application.

[0016] 2) The expression level of the hematological marker resistin in the present invention showed significant differences between COPD patients with sarcopenia and COPD patients with normal muscle function; the expression level of resistin was significantly correlated with the muscle function of COPD patients; ROC curve analysis showed that it had high specificity and sensitivity as a detection variable. Therefore, resistin can be used as a detection marker for the diagnosis of sarcopenia in COPD patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Relationship between serum resistin levels and clinical characteristics in patients with chronic obstructive pulmonary disease.

[0018] Figure 2 Relationship between serum resistin level and skeletal muscle function in patients with chronic obstructive pulmonary disease.

[0019] Figure 3 The clinical value of serum resistin in predicting sarcopenia in patients with chronic obstructive pulmonary disease. DETAILED DESCRIPTION

[0020] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will contribute to the understanding of the present invention, but these embodiments are only for the purpose of illustrating the present invention, and the present invention is not limited to these contents. The operating methods in the embodiments are all conventional operating methods in the art.

[0021] Example 1. Relationship between serum resistin levels and clinical characteristics in patients with COPD.

[0022] In order to verify the clinical value of resistin, blood samples were collected from patients with stable COPD sarcopenia and patients with stable COPD non-sarcopenia. After sampling, serum samples were separated and immediately stored at -80°C for testing. The expression level of resistin in each sample was detected using an ELISA kit (R&D Company; Catalog No.: DGD150). First, compared with patients with early COPD (GOLD 1 and 2), the expression of resistin in patients with late COPD (GOLD 3 and 4) increased ( Figure 1 A). Next, we analyzed the relationship between serum resistin level and lung function, and the results showed that serum resistin level was negatively correlated with the predicted forced expiratory volume in one second (FEV1% predicted; r = -0.193, p = 0.037) and the forced expiratory volume in one second / predicted forced vital capacity percentage (FEV1 / FVC, r = -0.206, p = 0.026; Figure 1 B). mMRC and CAT scores are widely used to evaluate the clinical symptoms of COPD patients. Figure 1 As shown in Figure C, serum resistin levels were positively correlated with mMRC (r = 0.288, p = 0.002) and CAT scores (r = 0.215, p = 0.023). Reduced exercise tolerance is one of the main clinical characteristics of patients with COPD and is associated with poor prognosis. 6MWD (6-minute walk distance) is a widely used tool to assess exercise tolerance. Figure 1As shown in D, serum resistin level was negatively correlated with 6MWD (r = -0.527, p < 0.001). In addition, malnutrition has a negative impact on lung function and increases disease exacerbations and mortality. Body mass index (BMI), body fat percentage, and fat-free body mass index (FFMI) are used to assess the nutritional status of COPD patients. Figure 1 As shown in Figure E, serum resistin levels were negatively correlated with BMI (r = -0.274, p = 0.003) and FFMI (r = -0.284, p = 0.014), but not with body fat percentage. Overall, these results suggest that resistin is an important biomarker for patients with COPD.

[0023] Example 2. Relationship between serum resistin level and skeletal muscle function in patients with COPD.

[0024] like Figure 2 As shown in A, serum resistin levels were negatively correlated with SMMI (skeletal muscle mass index; r = -0.273, p = 0.010). Next, we analyzed the relationship between resistin levels and muscle strength. Figure 2 As shown in B, serum resistin levels were negatively correlated with quadriceps muscle strength (QMS; r = -0.332, p = 0.004) and handgrip strength (HGS; r = -0.190, p = 0.040). Physical impairment is a common feature of COPD patients and is caused by poor skeletal muscle function. 5STS is used to assess physical performance; higher 5STS indicates lower physical performance. Figure 2 As shown in C, serum resistin levels were positively correlated with 5STS (r = 0.212, p = 0.047). Finally, we analyzed the relationship between resistin and rectus femoris. Abnormal lower limb muscle mass is a manifestation of skeletal muscle dysfunction in patients with COPD. Compared with healthy controls, the thickness and cross-sectional area of ​​the rectus femoris in patients with COPD were significantly reduced. Figure 2 D, Serum resistin levels were negatively correlated with Rfthick (r = -0.440, p < 0.001) and RFcsa (r = -0.445, p < 0.001). These findings suggest that resistin is an important skeletal muscle biomarker in patients with COPD.

[0025] Example 3. The clinical value of serum resistin in predicting sarcopenia in patients with COPD.

[0026] The serum resistin level in COPD patients with sarcopenia was significantly higher than that in COPD patients without sarcopenia ( Figure 3 A). In the training set ( Figure 3B), the sensitivity and specificity of serum resistin level in predicting sarcopenia were 93.02% and 62.16%, respectively (cutoff point was 7.138 ng / mL, AUC value was 0.828 (95% CI: 0.753-0.903)). We then verified the diagnostic efficacy of resistin in the validation set. Figure 3 As shown in C, the AUC value of predicting sarcopenia based on resistin was 0.818 (95% CI: 0.711-0.926, p<0.001).

[0027] Collectively, these results suggest that serum resistin expression may serve as a biomarker for predicting sarcopenia in COPD patients.

[0028] The experimental methods described in the above examples are as follows.

[0029] 1. Research subjects.

[0030] This was a prospective multicenter cross-sectional study. Patients with stable COPD were recruited from August 2018 to December 2019 at the First Affiliated Hospital of China Medical University and the First Affiliated Hospital of Dalian Medical University.

[0031] The inclusion criteria were as follows: stable COPD diagnosed according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria; age ≥40 and <80.

[0032] Exclusion criteria were as follows: 1. COPD exacerbation within the last month; 2. Comorbidity with severe cardiovascular disease and active lung disease; 3. Long-term use of systemic steroids; 4. Unable to read or understand the informed consent form. This study was approved by the Research Ethics Committee of the First Hospital of China Medical University and the Ethics Committee of the First Hospital of Dalian Medical University.

[0033] A total of 117 COPD patients recruited from the First Hospital of China Medical University were included in the development set to evaluate the clinical value of resistin and determine the cutoff value. A total of 118 stable COPD patients recruited from the First Hospital of Dalian Medical University were included in the validation set.

[0034] 2. Questionnaire scores for COPD patients.

[0035] The Chinese version of the modified British Medical Research Council (mMRC) Dyspnea Index was used to assess dyspnea symptoms, and the Chinese version of the COPD Assessment Test (CAT) was used to assess health status.

[0036] Table 1. Modified British Medical Research Association Dyspnea Index Scoring Table

[0037] .

[0038] Table 2. COPD Assessment Test Scoring Sheet

[0039] .

[0040] 3. Quadriceps muscle strength measurement.

[0041] Quadriceps strength (QMS) in COPD patients was measured using a dynamometer, following the instructions provided by the manufacturer and previous studies. The specific method was as follows: the subject's knee was bent 90° and the dynamometer was placed immediately afterwards. The tip of the dynamometer was located 5 cm proximal to the lateral calf bone on the anterior surface of the leg, perpendicular to the long axis of the tibia. The medical staff held the dynamometer plate and applied force, while instructing the subject to push the dynamometer plate with the knee extended as much as possible and keep it in the same position for 4 seconds. The same steps were repeated twice, with an interval of 30-60 seconds between each time. The average of the two assessments on each side of the lower limb was recorded as the maximum unilateral contraction force. Then, the average of the bilateral contraction force was the quadriceps strength.

[0042] 4. Fat-free mass index.

[0043] The fat-free mass index (FFMI) of the subjects was measured by bioelectrical impedance meter. The calculation method of FFMI is as follows: FFMI = weight (kg) × (1-body fat percentage) / height (m) 2 .

[0044] 5. Sit-stand experiment.

[0045] In this study, the sit-to-stand test (STST) used two modes: a five-repetition sit-to-stand test (5STS) and a 30-second sit-to-stand test (30STS). The specific method is as follows: the subject sits on a 48 cm high chair without armrests, with both feet on the ground, the back supported by the back of the chair, and the hands clasped in front of the chest. After hearing the test start instruction from the medical staff, the subject stood up and sat down as quickly as possible for 5 times. During the test, the subject kept his arms crossed in front of his chest and the knee joints were fully straight when standing. The medical staff verbally encouraged the subject during the test and recorded the time. The five-repetition sit-to-stand test was repeated three times with a 1-minute rest period in between. The average of the three tests was recorded as the result. For the 30-second sit-to-stand test, the medical staff recorded the number of times the subject stood up and sat down within 30 seconds. Similarly, the test was repeated three times, and the average value was recorded as the result.

[0046] 6.6MWT.

[0047] Follow the American Thoracic Society's guidelines for the 6-minute walk test. First, choose an enclosed, long, and straight indoor corridor that is 30 meters long. Before the test, explain the test method to the patient in detail and tell the patient to walk as far as possible. If the patient feels short of breath or has chest pain or dizziness, allow the patient to slow down or stop to rest. If the above symptoms worsen or cannot be relieved after rest, the test should be stopped immediately. During the test, use standardized language to encourage the patient. After 6 minutes, when the patient hears the experimenter's instruction "time is up", he or she stops walking immediately. The experimenter records the distance walked by the patient in meters, which is recorded as 6MWD.

[0048] 7. Ultrasound measurement of rectus femoris thickness and cross-sectional area.

[0049] The thickness and cross-sectional area of ​​the rectus femoris muscle were measured as follows. Grayscale ultrasound scans were performed using the Aixplorer ultrasound scanning system and a 4-15-MHz linear array transducer (5 cm width). The subjects rested quietly for 15 minutes before the test, then lay supine on the examination table and relaxed all muscles. Our ultrasound doctors were formally trained and had more than 10 years of experience. In order to reduce muscle deformation caused by external forces, we set up a bracket to fix the ultrasound probe and place it perpendicular to the patient's dominant leg. The transducer was perpendicular to the long axis of the dominant leg and accurately positioned at 3 / 5 of the distance from the anterior superior iliac spine to the upper edge of the patella. This is the highest point of the thigh. The entire cross-section of the rectus femoris muscle of all subjects can be seen in the same field of view of the Aixplorer ultrasound scanning system, and other muscles of the quadriceps group cannot be included. The scanning depth was set to detect the direction of the femur. Before image acquisition, gentle contraction-relaxation maneuvers were used to delineate the muscle intervals. The thickness of the rectus femoris (RF) was calculated by outlining the medial echogenic line of the rectus femoris on the frozen image with a moving cursor. thick The cross-sectional area of ​​the rectus femoris, RF csa ). The average of three measurements with an error within 10% is taken as RF thick and RF csa , and record it.

[0050] 8. Assessment of sarcopenia.

[0051] The definition of sarcopenia follows the guidelines of the Asian Sarcopenia Working Group as follows: low muscle mass (bioelectrical impedance [male, <7.0 kg / m 2 ; Women, <5.7 kg / m 2 ]); low muscle strength (grip strength [males, <28 kg; females <18 kg]); and poor physical performance (5-repetition chair stand test [5STS], ≥12 seconds).

[0052] Muscle mass was assessed using bioelectrical impedance analysis [BIA] (InBody770; InBody). Muscle strength was assessed by grip strength using a JAMAR® Plus+ hand dynamometer (Sammons Preston, Bolingbrook, IL, USA). For body composition assessment, patients stood barefoot on the platform of the InBody with the soles of their feet placed on the electrodes. They grasped the handles of the device with their thumbs and fingers to maintain direct contact with the electrodes and remained still for approximately 1 min while keeping their elbows fully extended and their shoulder joints abducted at approximately 30°. Grip strength (HGS) assessment was performed using standard technique with the patient in a seated position with the elbows flexed at a 90° angle and the wrists in a neutral position. Subjects were instructed to apply maximal HGS 3 times with both hands, allowing 30 seconds of rest between each measurement.

[0053] 9. Enzyme-linked immunosorbent assay (ELISA).

[0054] Venous blood was collected in a fasting state. The blood samples were collected into serum separator tubes, left to clot at room temperature for 2 h, and then centrifuged at 3000 rpm for 15 min at room temperature. The supernatant was obtained and stored in a -80 °C refrigerator. The expression level of resistin was assessed by a high-sensitivity ELISA kit (R&D) according to the manufacturer's instructions.

Claims

1. Use of a reagent for detecting the protein expression level of resistin in serum samples in the preparation of an auxiliary diagnostic product for chronic obstructive pulmonary disease combined with sarcopenia; the auxiliary diagnostic product for chronic obstructive pulmonary disease combined with sarcopenia uses resistin protein as a marker; the expression level of resistin protein is positively correlated with the incidence of chronic obstructive pulmonary disease combined with sarcopenia.

2. The use according to claim 1, characterized in that: The product detects the expression level of resistin in serum samples through a chip or enzyme-linked immunosorbent assay.

3. The use according to claim 1, characterized in that: The product contains antibodies that specifically bind to resistin.

4. The use according to claim 1, characterized in that: The product is a chip, a preparation or a kit.

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