Effect of Low Molecular Weight Heparin Calcium Combined with Argatroban on the Treatment, Vascular Endothelial Function, Inflammatory Factors and Serum Neurological Function in Patients with Acute Cerebral Infarction

Xiaoli Yao1, Xiaofang Yue2, Yue Yang3 and Guobin Zhang3,* 1Department of Neurology, Zhengzhou Central Hospital Affiliated to Zhengzhou University, Zhengzhou, 450000, China 2Department of Neurology, Shanghai Sixth People’s Hospital East Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai, 201306, China 3Department of Radiology, Shanghai Sixth People’s Hospital East Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai, 201306, China Article Information Received 17 November 2020 Revised 01 March 2021 Accepted 18 April 2021 Available online 31 January 2022 (early access)


INTRODUCTION
A cute cerebral infarction (ACI), also known as cerebral infarction, refers to ischemic stroke in the brain. As a common neurological disease in the clinic, it accounts for more than 60% of cerebral infarction cases, which has an acute onset and rapid progress. Its clinical manifestations include facial paralysis, upper extremity hemiplegia, etc. In severe cases, diplopia, dizziness and arrhythmia will occur, even threatening life (Sun et al., 2019;. In the past, ACI mostly occurred in the middleaged and elderly population. Recent years witnessed its occurrence in younger population. It can occur at any age with no specific morbidity group, and there is no forewarning at the time of onset. Therefore, effective treatment drugs and methods are the key to protecting patients' life and health (Wu et al., 2016). Clinical studies have shown that anticoagulant therapy has a good effect in preventing prolongation of thrombus, recurrence of stroke, secondary thrombosis in distal small vessels, and improving collateral microcirculation (Peng and Wu, 2018). At present, studies have confirmed that argatroban and low-molecular-weight heparin (LMWH) have a significant therapeutic effect on ACI, but combined use of the two drugs has not been evaluated Chen et al., 2018;Qiu et al., 2021). Therefore, this study investigates the actual efficacy of low-molecular-weight heparin calcium (LMWHC) combined with argatroban in the treatment of ACI, and determines the basic mechanism of the drug by detecting changes in inflammatory factors, vascular endothelial function, and neural function before and after treatment, and ultimately provides data reference for the clinic.

General information
Inclusion criteria: ACI diagnosis was performed according to the standards in the 2018 Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke (Peng and Wu, 2018), and only a single infarct was confirmed by MRI or CT, which had consistent located signs, with no cerebral hemorrhage, brain occupation. The first onset or recurrence left no obvious sequelae. The disease attacked within 3 days, and no thrombolytic drug therapy was used. NIHSS score was 2～15. There was no history of allergy to argatroban, and no statins, immune or inflammation inhibitors were taken within 4 weeks before the treatment.
Exclusion criteria: those younger than 18 years old or older than 85 years old, those accompanied with immune system diseases, cancer, hemorrhagic diseases or abnormal liver and kidney functions, as well as pregnant or lactating women.
Case selection: According to the above criteria, 80 patients who received ACI treatment at the First Hospital of XX City from January 2018 to December 2020 were selected, who were divided into two groups by random table method-treatment group (LMWHC + argatroban + conventional treatment) and control group (conventional treatment). The treatment lasted 7 days. The clinical efficacy, changes in inflammatory factors (hs-CRP, TNF-α, IL-6, IL-8, MMP-9, Lp-PLA2), vascular endothelial function indicators (NO, ET-1, FMD), neurological impairment (NIHSS score) were compared between the two groups at 14 days after treatment. The requirements for blood collection were that patients should not eat highfat, high-tyramine foods for the first 3 days, start fasting at 12h before blood collection. 8mL of anticoagulant blood was collected in the morning on an empty stomach before treatment and on the 14th day after treatment, which was centrifuged at 4°C 3000 r/min for 10 min to separate plasma and then stored at -70°C for future inspection.

Treatment method
For treatment group, the dosage was calculated according to body weight, and 3000～5000AU LMWHC (Shenzhen Sciprogen Bio-Pharmaceutical Co., Ltd., National Medicine Permission Number H20060190) was injected subcutaneously, twice per person per day, with an interval of 12h for 7 days. At the same time, on the first and second days of treatment, continuous intravenous pumping of 60mg argatroban (Tianjin Institute of Pharmaceutical Research Co., Ltd., approval number: National Medicine Permission Number H20050918) was given for 24 h per day per person without interruption; From the 3rd to 7th days of treatment, intravenous infusion of 10mg argatroban was given, 2 times a day and 3 h per time.
For control group, when giving conventional treatment, enteric-coated aspirin (0.1g) was taken orally every night, intravenous drip of edaravone injection (30mg+9g/L sodium chloride injection 250mL, twice a day) and Danshen ligustrazin for injection (10mL+50g/L glucose injection 250mL, once a day) was given, and 10mg atorvastatin calcium was orally taken once a day after supper. In addition, according to the state of illness and complications, increase the amount of other drugs appropriately for symptomatic treatment and maintain water and electrolyte balance.

Detection of neurological function factors
ELISA method (with reagent purchased from Shanghai Keshun Biotechnology Co., Ltd.) was used to detect copeptin, N-terminal pro-brain natriuretic peptide (NT-proBNP), polyamine oxidase (PAO), S -100B protein before and after the treatment.

Determination of cerebral blood flow
The average blood flow velocity of the middle cerebral artery (MCA) and the anterior cerebral artery (ACA) was evaluated according to the cranial Doppler (TCD) detection results of the cerebral blood flow status before and after treatment.

Detection of vascular endothelial function
Nitric oxide (NO) was detected by nitrate reductase (purchased from Nanjing Jiancheng Bio-Engineering Institute). Endothelin-1 (ET-1) was detected by radioimmunoassay (purchased from East Asia Institute of Immunology, General Hospital of the People's Liberation Army). When testing the endothelium-dependent vasodilation function (FMD) mediated by brachial artery blood flow, the color ultrasound diagnostic instrument (HP 5500, USA) was used to detect vasodilator drugs O n l i n e

F i r s t A r t i c l e
discontinued 12 h ago. The patient needs to lie supine 10 min before measurement of basic diameter of the brachial artery (d 0 ); when measuring the inner diameter of reactive hyperemia induced brachial artery (d 1 ), place the sphygmomanometer in the cuff on the distal end of the target artery, inflate and pressurize to 300mmHg, deflate at 4min, and read the result between 60～90s. FMD calculation formula:

Statistical analysis
SPS S16.0 statistical analysis software was used to compare the data of each group. When the measurement data conformed to normal distribution, (x ̅ ±s) was used for χ 2 test; when the measurement data were not normally distributed, the median and inter-quartile range was compared. The difference was statistically significant when P<0.05.

RESULTS
There was no statistically significant difference in comparison of age, gender, underlying disease (hypertension, diabetes), and cerebral infarction site between the groups (P>0.05) ( Table I).  Table II shows the effect of LMWHC and argatroban on score on neurological deficit (NIHSS), cerebral blood flow (MCA, ACA), neurological function indicators (copeptin, NT-proBNP, PAO, S-100B ET-1), vascular endothelial function (NO, ET-1, FMD) and Table II

O n l i n e F i r s t A r t i c l e
groups showed insignificant differences between the two groups. After treatment with LMWHC+argatroban, all these parameters show statistically significant differences. The NIHSS of the treatment group, on the 14th day after treatment shows statistically significant difference between the groups (P<0.05). The total effective rate was 97.01% in the treatment group after treatment compared to 80.32% in the control group, showing significant difference (P<0.05).
The cerebral blood flow indicators (MCA, ACA) and NO and FMD as vascular endothelial function shows significant increase after treatment with LMWHC and argatroban. On the other hand all neurological indicators (copeptin, NT-proBNP, PAO, S-100B ET-1), ET-1 as endothelial function and all inflammatory factors showed significant decrease after treatment with LMWHC and argatroban.

DISCUSSION
Currently, anticoagulant and antiplatelet aggregation drugs are common clinical drugs for infarction treatment, such as low-molecular-weight heparin, which has the characteristics of long biological half-life, low bleeding risk, and good safety. It does not affect vascular permeability when it exists in the form of calcium salt, and can play a role in improving cerebral hemodynamics and producing antithrombotic effects, but it is not ideal for the treatment of progressive cerebral infarction when used alone, so its combined use with other anticoagulant drugs in clinical application is currently the main treatment strategy (Arahata and Asakura, 2018;Zhu et al., 2018). According to literature reports, low-molecular-weight heparin calcium combined with urokinase can effectively produce antithrombotic, anticoagulant effects and improve whole blood viscosity, thus significantly improving the overall efficacy of cerebral infarction (Huang and Pan, 2012); when vinpocetine is combined with LMWHC for the treatment of senile chronic cerebral circulatory insufficiency, clinical symptoms can be significantly improved, blood rheology and viscosity indicators have improved significantly without any negative effects (Fatima et al., 2020). LMWH therapy combined with clopidogrel hydrogen sulfate has significant effect in treating progressive cerebral infarction, which produces better effect than use of LMWH alone and significantly improves the patient's cerebral hemodynamics. Moreover, the risk of adverse reactions has not increased (Arahata and Asakura, 2018).
Argatroban is used as a thrombin inhibitor (with molecular weight 527Da), which can reversibly bind to the active site of thrombin. After binding, it can inactivate thrombin to alleviate the patient's circulating hypercoagulable state and avoid the formation of secondary embolism. It is highly selective and does not require aid of antithrombin to produce anticoagulant effect (Yi-Cui et al., 2015;Mohamed and Coombe, 2017). Argatroban has good safety in clinical use. The therapeutic dose does not interfere with platelet function, nor will it cause bleeding. At the same time, it is not immunogenic and does not have cross immunoreaction with heparin-induced antibodies. The metabolic pathway of argatroban is liver, so those with renal insufficiency do not need to reduce the dosage. Moreover, with short half-life and fast onset, it can regulate vascular endothelial cell function and reduce inflammation (Ishibashi et al., 2013). According to literature reports, when argatroban is used to treat ACI, the damage to vascular endothelial function can be reduced and the inflammatory response can be significantly reduced, so the patient's nerve function is improved, showing significantly improved therapeutic effect (Xu et al., 2017). In addition, edaravone combined with argatroban for the treatment of ACI can also significantly improve the patient's nerve function and body hypercoagulability by reducing the level of inflammatory factors .
The thrombus formed by fibrin contains a lot of thrombin, which will be released when the thrombus is dissolved, thereby causing reformation of the thrombus. LMWHC has a molecular weight 10 times bigger than that of argatroban, and it cannot exert antagonistic effect on bound thrombin. However, because of its small molecular weight, argatroban can inactivate thrombin inside the thrombus, thus producing good thrombolytic effect (Denorme et al., 2016). By contrast, LMWHC also has its own advantages, such as longer half-life, anticoagulation and fibrinolytic effects. Related studies have reported that LMWHC, argatroban produce unobvious clinical efficacy in the treatment of acute cerebral infarction. Therefore, this study clinically combines LMWHC and argatroban for the first time, and has achieved ideal treatment effect (Hirsh and Raschke, 2004;Zhang et al., 2020).
This study compares the clinical efficacy (NIHSS score) of acute cerebral infarction, TCD cerebral blood flow, serum neurological function indicators (copeptin, NT-proBNP, PAO, S-100B), changes in inflammatory factors (hs-CRP, TNF-α, IL-6, IL-8, MMP-9, Lp-PLA2) and vascular endothelial function indicators (NO, ET-1, FMD) between combined medication and conventional therapy. The results indicate that on the 14th day after treatment, the treatment group has significantly higher total effective rate than the control group, showing significant differences in vascular endothelial function, inflammatory factor levels, serum neurological function indicators and NIHSS between the two groups (P<0.05). Moreover, no O n l i n e

F i r s t A r t i c l e
Effects of LMWHC in Patients with Acute Cerebral Infarction clinical adverse reactions associated with argatroban and LMWHC was found during treatment (Yang et al., 2020). The indicators tested in this study, such as MCA and ACA, can reflect blood supply condition of brain tissue. If the value decreases, it indicates ischemia and hypoxia of brain tissue in the infarct area, suggesting severe brain function damage. Concentration of copeptin, NT-proBNP, PAO and S-100B in serum reflects the functional status of brain tissue, which is also a common clinical evaluation item. Vascular endothelial function damage and inflammation will promote the occurrence of ACI. Where, NO, ET-1 and FMD are typical endothelial function indicators, hs-CRP, TNF-α, IL-6, IL-8, MMP-9 and Lp-PLA2 are the most reported inflammatory markers in clinic. The detection of the above indicators features simple sampling and strong representativeness, which can clarify the basic intervention mechanism of therapeutic drugs on ACI patients. ACI patients mainly manifest neurological damage. The results of this study indicate that the combined application of LMWHC and argatroban not only plays an anticoagulant role in treatment, but also can significantly improve vascular endothelial function and antagonize the inflammatory response, thereby restoring the patient's neurological function (Wu et al., 2020;Gao et al., 2018).
To conclude, the combination of LMWHC and argatroban for clinical treatment of ACI can improve patients' condition in overall and protect patients' brain tissue function, which means practical significance for improving surgical treatment effect and reducing postoperative complications in clinical application .