Effect of oral ligustrazine phosphate with cerebroside carnosine on neurological function and serum inflammatory factors among patients with ischemic cerebrovascular disease: a quasi-experimental study

Abstract

Objectives

To explore the effect of ligustrazine phosphate tablets combined with cerebroside carnosine on neurological function and serum inflammatory factors among patients with ischemic cerebrovascular disease.

Methods

This was a quasi-experimental study. From March 2019 to February 2022, 126 patients were non-randomly divided into control (n = 63) and intervention (n = 63) groups. The control group received routine treatment such as encephaloside and carnosine, while the intervention group was treated with ligustrazine phosphate tablets and brain glycoside carnosine for 2 weeks. Serum S-100β protein, nerve growth factor (NGF), inflammatory factors [tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP)], National Institutes of Health Stroke scale (NIHSS) and and Activities of Daily Living (ADL) scale were assessed before and after the intervention.

Results

After treatment, the level of S-100β in the intervention group was lower, and the level of NGF was significantly higher than in the control group. The levels of TNF-α and CRP in the intervention group were reduced. The NIHSS score of the intervention group was lower, and the ADL score was significantly higher than that of the control group.

Conclusions

The combination of ligustrazine phosphate tablets with cerebroside carnosine can improve neurological function, alleviate inflammation, and enhance the quality of life, worthy of clinical promotion.

Introduction

Ischemic cerebrovascular illness is a common illness in the department of neurology, and it is the general name of different degrees of ischemic cerebrovascular illness. Its clinical manifestations and severity vary greatly due to different cerebrovascular involvements. The common symptoms are vertigo, consciousness and language disorder, unilateral facial numbness, limb numbness and so on [1,2,3]. Ischemic stroke is the main clinical type, with the highest incidence, disability, recurrence and mortality in ischemic cerebrovascular illness [4]. Mild degree ischemic cerebrovascular illness is a transient ischemic attack caused by ischemia. Although it can be fully recovered within 24 h, it suggests that the risk of stroke is very high in the near future, and it is necessary to seek medical advice in time to prevent sudden change of the illness [5, 6]. Epidemiological studies have shown that ischemic cerebrovascular illness mostly occurs in the middle-aged and elderly in China, which tends to increase year by year, and the age of first onset is getting younger and younger, and ischemic stroke is one of the important causes of disability and death of Chinese residents. Serious harm to people's physical and mental health, the prevention and remedy of ischemic cerebrovascular illness is urgent [7, 8]. One of the factors that contribute to the progression and severity of ischemic cerebrovascular diseases is inflammation. In patients with cerebral ischemia, both systemic and neuroinflammation can exacerbate tissue damage and negatively affect neurological function [9,10,11]. Therefore, managing inflammation as a therapeutic strategy may help reduce neural damage and improve treatment outcomes.

In this context, various pharmaceutical compounds have been investigated to reduce inflammation and improve neurological function. Ligustrazine phosphate, a plant-derived compound with anti-inflammatory and neuroprotective properties, has gained particular attention in the treatment of neurological and ischemic cerebrovascular diseases.

Ligosterazine injection has been widely used in China for the treatment of acute ischemic stroke (ACI) and coronary artery disease in the past decades [12]. Ligustrazine is the main active ingredient of the plant Ligusticum chuanxiong, an bioactive alkaloid extracted from Ligusticum Chuanxiong Hort [11]. In recent years, ligustrazine has gained popularity in the treatment of ischemic cerebrovascular diseases. Numerous studies have reported that ligustrazine has a protective effect against stroke-related diseases, such as reducing blood–brain barrier permeability, dilating cerebral vessels, preventing thrombosis, providing anti-inflammatory and antioxidant effects, and activating microglia. Activated microglia increase the production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), leading to a severe neuroinflammatory response. Recent studies have shown that ligustrazine prevents apoptosis of PC12 neurons via Bcl-2 protein and caspase-3. However, the molecular targets and mechanisms of ligustrazine’s neuroprotective function remain unclear. Therefore, understanding the mechanisms of ligustrazine in ischemic stroke is critical [9, 10]. Carnosine is a dipeptide composed of β-alanine and L-histidine, known as an exercise enhancer, and is primarily found in skeletal muscles, with smaller amounts in the heart muscle and brain. This compound can help optimize energy metabolism, improve mitochondrial function, and reduce systemic inflammation and oxidative stress. Carnosine is capable of crossing the blood–brain barrier and is synthesized in olfactory neurons and glial cells, particularly in mature oligodendrocytes. Furthermore, carnosine and homocarnosine (its analog in the brain) are present in cerebrospinal fluid and act as endogenous antioxidants, neuromodulators, and neuroprotective molecules [10].

The purpose of clinical remedy is to save lives, reduce disability, prevent recurrence and improve the quality of life of sufferers. Brain glycosides carnosine can improve the cerebral circulation by participating in the growth, differentiation and regeneration of brain tissue neurons and promoting the metabolism of brain tissue. So far, good results have been achieved [13]. The determination of S-100 β has important clinical value in the remedy of cerebrovascular illnesses, the diagnosis of brain injury and the severity of brain injury, as well as in judging the prognosis. The higher its level is, the more serious the illness is, indicating a poor prognosis of the illness. After the occurrence of acute stroke, the concentration of S100-β protein increased significantly at 8 ~ 24 h [14, 15]. And also TNF-α and CRP are both important indicators of inflammatory response. The contents of TNF-α and CPR increased, and then decreased after the inflammatory reaction was alleviated [16]. This research is of great importance, because ischemic cerebrovascular diseases remain one of the leading causes of death and disability worldwide, and improving treatment methods for these patients is a medical priority. Currently, available treatments mainly focus on symptom management and preventing further damage, but their long-term effectiveness is limited. Given the significant role of inflammation in the pathogenesis of ischemic cerebrovascular diseases, identifying and evaluating new treatments that can reduce inflammation while improving neurological function is essential. The combination of ligustrazine phosphate with cerebroside carnosine may offer an innovative and effective approach to reduce neurological and inflammatory damage. This study could contribute to advancements in medical treatments and improve clinical outcomes for patients with ischemic cerebrovascular diseases, ultimately providing a path for the development of more effective combination therapies.

Materials and methods

Study design, setting, and participants

This was a quasi-experimental study. This study was carried out after being examined by the Medical Ethics Committee of our hospital. Inclusion criteria: (i) in accordance with the diagnostic criteria of ischemic cerebrovascular illness in the book of "Cerebrovascular illness/Transient Ischemic Attack" [17]; (ii) normal liver and kidney function; (iii) informed consent signed by the sufferer's family or himself; and (iv) good sufferer compliance, the sufferers were conscious.

Exclusion criteria: (i) previous hypersensitivity to ligustrazine phosphate tablets or cerebroside carnosine drugs; (ii) sufferers with important organ failure; (iii) sufferers with malignant tumor; (iv) abnormal blood system or immune system; and (v) previous craniotomy. Patients who received additional treatments such as acupuncture, complementary medicine, specific exercise programs, or special dietary regimens were excluded from the study.

From March 2019 to February 2022, patients with ischemic cerebrovascular disease who admitted into our hospital were opted as subjects. In this quasi-experimental study, a total of 126 patients with ischemic cerebrovascular disease who met the inclusion criteria were enrolled. To ensure an adequate balance of baseline characteristics between the intervention and control groups, patients were first stratified into different layers based on key clinical characteristics, such as age, gender, and disease type. After stratifying the patients into these initial layers, they were non-randomly assigned to either the intervention or control group. In general, each layer of patients contained a similar number of individuals in both groups to minimize baseline clinical differences between the groups.

Power analysis calculations with G*Power software indicate that (power = 80%, α error probability = 0.05, number of groups = 2, and number of measurements = 2) 124 participants would be needed to detect an effect size of 0.45. Finally, a total of 63 patients were assigned to each group. During the study period, no discontinuation or drop-out occurred in both groups.

Data collection and intervention

Both groups were instructed to follow a low salt and low-fat diet, maintain good rest and mood, and regularly monitor their blood pressure and blood glucose. In addition, both groups were provided with conventional antiplatelet and anticoagulant medications to enhance brain circulation, as well as various statins based on the type and severity of the condition [18]. The control group received intravenous cerebroside carnosine (Manufacturer: Jilin Sihuan Pharmaceutical Co., Ltd.; Approval number: GYZZ H22025047; Specification: 5 ml). The cerebroside carnosine (10 mL) was added to 250 mL of a 0.9% sodium chloride solution and administered via slow drip once a day for 2 weeks. In contrast, the intervention group received the same intravenous cerebroside carnosine treatment as the control group. However, in addition to the intravenous infusion, the intervention group was also treated with oral Ligustrazine Phosphate tablets (Manufacturer: Guangzhou Kanghe Pharmaceutical Co., Ltd.; Approval number: H44024272; Specification: 50 mg * 100 tablets). These tablets were taken twice daily, with two tablets per dose, and the treatment lasted for 2 weeks [19].

Measures

Neurological function index: Two tubes of 5 mL venous blood were drawn from the two groups before and after intervention, and the blood was centrifuged for about 20 min (2000–3000 rpm/min). After that, the upper serum was collected and the content of S-100β protein in serum was quantitatively detected by fluorescence quantitative immunochromatography. The kit was provided by Wuhan Mingde Biotechnology Co., Ltd. The content of nerve growth factor (NGF) was determined by double antibody sandwich method. The kit was provided by Beijing baiaolaibo Technology Co., Ltd [20, 21].

Serum inflammatory factors: Fasting elbow venous blood 4 mL was collected before and after intervention in both groups. After successful centrifugation, the supernatant was taken out, and the levels of tumor necrosis factor-α (TNF-α) and C-reactive protein (CRP) [22] in the two groups were determined by enzyme-linked immunosorbent assay (ELISA). The kit was provided by Shanghai Guangrui Biotechnology Co., Ltd.

National Institutes of Health Stroke scale (NIHSS) score and Activities of Daily Living (ADL) score [23, 24]: NIHSS was used to evaluate the neurological status of sufferers with ischemic cerebrovascular illness. It includes 11 items, such as consciousness, gaze, language, muscle strength of upper and lessened extremities and so on. The score range was 0 ~ 42, and the higher the score, the more severe the neurological function injury. The ADL scale was evaluated the quality of life of sufferers by their dependence on the most basic daily activities, such as bathing, dressing, and defecation. The highest score was 100, and the score of 60 indicated that the sufferer basically took care of himself, only mild dysfunction, and some daily activities can be completed independently. The score among 40 and 60 meant that most of the sufferers' daily activities needed external assistance, 40 or less indicated that they had severe dysfunction, most of their daily activities can not be completed by themselves, and relied on others for help. The lower the score, the stronger the dependence.

Data analysis

SPSS22.0 statistical software was used to analyze the data of this study. The observation indicators selected in this study were expressed by the average ± standard deviation ($\overline{x}$ ± s), using t test. When the P < 0.05, it indicated that the discrepancy was significant and comparable, otherwise it was incomparable.

Results

In this study, all 126 patients completed the trial and adhered to the assigned treatment protocol until the end of the study, with no dropouts reported. Adherence to the treatment regimen was monitored throughout the study, and there were no significant deviations or non-compliance observed. In addition, no serious adverse events or unexpected side effects were reported during the course of the study. The treatment was well-tolerated by all participants, and no adverse events were deemed to be related to the interventions.

In the control group, there were 29 males and 34 females, aged 56–73 years, with an average age of (62.81 ± 7.26) years, and the illness types included transient ischemic attack (n = 9), stroke (n = 42) and other types (n = 12). In the intervention group, there were 32 males and 31 females, aged 57–74 years, the average age was (63.34 ± 7.58) years, and the illness types included transient ischemic attack (n = 7), stroke (n = 43) and other types (n = 13). There were no statistical differences between the two groups regarding these variables (P > 0.05) (Table 1).

Table 1 Comparison of demographic characteristics between two groups

After intervention, the content of S-100 β in the intervention group and the control group was lessened than that before intervention, whereas the content of NGF in both groups was boosted (P < 0.05). After intervention, the content of S-100 β in the intervention group was lessened than that in the control group (P < 0.05). The content of NGF in the intervention group was significantly strengthened than that in the control group after intervention (P < 0.05) (Table 2).

Table 2 Comparison of S-100β and NGF between the two groups$\left( {\overline{x}\, \pm \,s} \right)$

After intervention, TNF-α and CRP levels in the intervention group and the control group were lessened than those before intervention (P < 0.05), and TNF-α and CRP levels in the intervention group were lessened than those in the control group (P < 0.05) (Table 3).

Table 3 Comparison of TNF-α and CRP between the two groups $\left( {\overline{x}\, \pm \,s} \right)$

After intervention, the NIHSS scores of the patients in the intervention group and the control group were lessened than those before the intervention (P < 0.05), and the ADL scores of the two groups were boosted than those before intervention (P < 0.05). The NIHSS score of the intervention group was lessened than that of the control group after the intervention (P < 0.05), and the ADL score of the intervention group was significantly strengthened than that of the control group (P < 0.05) (Table 4).

Table 4 Comparison of National Institutes of Health Stroke scale (NIHSS) and activities of Daily living (ADL) scores between the two groups (scores, $\overline{x}\, \pm \,s$)

Discussion

Ischemic cerebrovascular illness is caused by degeneration, necrosis or transient loss of function due to the disturbance of blood circulation in local brain tissue. Ischemic cerebrovascular illness is caused by a variety of causes, the most common atherosclerosis and hypertensive sclerosis caused by vascular wall damage. In addition, changes in blood composition and hemorheology lead to abnormal blood coagulation and indirectly lead to cerebral ischemia [25,26,27,28,29]. Previous study has shown that basic chronic illness such as hypertension, diabetes and hyperlipidemia are high risk factors for ischemic cerebrovascular illness [30,31,32]. In clinic, drug therapy is the main intervention means to strive to quickly recover the ischemia and hypoxia of brain tissue, promote the repair of nerve cells in the later stage, and carry out early rehabilitation remedy. In traditional Chinese medicine, tetramethyl pyrazine phosphate tablets have the effect of activating blood circulation, removing blood stasis and dredging collaterals. Modern pharmacological research shows that tetra methylpyrazine phosphate tablets have the effect of anti-platelet aggregation and depolymerization, so as to reduce blood viscosity, expand arterioles, improve microcirculation and intracranial blood supply. The determination of S-100 β has important clinical value in the remedy of cerebrovascular illnesses, the diagnosis of brain injury and the severity of brain injury, as well as in judging the prognosis. The higher its level is, the more serious the illness is, indicating a poor prognosis of the illness. After the occurrence of acute stroke, the concentration of S100-β protein increased significantly at 8 ~ 24 h [14, 15]. NGF can nourish and protect nerves, promote the re-growth and repair of central neurons, and accelerate the recovery of nervous system injury. The results of this study showed that the content of S-100 β in the test subgroup after remedy was lessened than that in the control subgroup, and the content of NGF in the test subgroup was memorably strengthened than that in the control subgroup, indicating that ligustrazine phosphate tablets linked with cerebroside carnosine in the remedy of sufferers with ischemic cerebrovascular illness can promote the recovery of nervous system and significantly improve neurological function. TNF- α and CRP are both important indicators of inflammatory response. The contents of TNF- α and CPR increased, and then decreased after the inflammatory reaction was alleviated [16]. The findings of this study highlight the potential benefits of combining ligustrazine phosphate with cerebroside carnosine in improving neurological function and reducing inflammation among patients with ischemic cerebrovascular disease. Our results align with previous research indicating that ligustrazine has neuroprotective properties and can significantly contribute to stroke recovery by reducing neuroinflammation and oxidative stress [12]. In addition, cerebroside carnosine has been shown to enhance mitochondrial function and optimize energy metabolism, which is critical for neuronal survival and function [9].

The significant decrease in serum S-100β protein levels observed in the intervention group suggests reduced neuronal damage compared to the control group. S-100β is a widely recognized biomarker of brain injury, and its reduction may indicate a protective effect of ligustrazine phosphate in mitigating ischemic damage. Furthermore, the observed increase in nerve growth factor (NGF) levels in the intervention group underscores the neurotrophic potential of the treatment, which is essential for neural repair and functional recovery.

Inflammation is a key contributor to the progression and severity of ischemic cerebrovascular disease. The significant reduction in TNF-α and CRP levels in the intervention group compared to the control group further supports the anti-inflammatory effects of ligustrazine phosphate. These findings are consistent with previous studies showing that ligosterazine reduces the expression of proinflammatory cytokines and modulates the circulation against ischemia/reperfusion injury [9]. Studies have shown that the inflammatory response involved in many inflammatory factors, such as TNF-α and CRP, is closely related to the progression of ischemic stroke. The decrease of the level of inflammatory factors indicates that the scope of brain injury is reduced [33, 34].

This study also demonstrated a substantial improvement in neurological function and daily living activities in patients receiving the combined therapy. The significant reduction in NIHSS scores and increase in ADL scores in the intervention group suggest enhanced recovery and quality of life. Previous meta-analyses have also highlighted the efficacy of ligustrazine in improving stroke outcomes by preventing thrombosis, reducing blood–brain barrier permeability, and promoting microglial activation [10].

This study has several limitations that should be acknowledged. First, the lack of randomization may introduce selection bias, potentially affecting the generalizability of our findings. Without random allocation, differences between groups could be influenced by confounding variables rather than the intervention itself. Second, the absence of blinding may have introduced observer bias, as both researchers and participants were aware of the treatment assignments, which could have influenced assessments and subjective outcomes. In addition, our sample size was relatively small, limiting the statistical power to detect subtle differences and increasing the risk of type II errors. Larger studies with more diverse populations are needed to confirm the robustness of our results. Finally, while this study provides valuable insights, it remains an observational analysis; thus, causal relationships cannot be firmly established. Future research should focus on conducting well-powered, randomized controlled trials with blinding to strengthen the evidence supporting our findings. Another limitation of this study is the short treatment duration of only 2 weeks, which may not fully capture the long-term effects of the therapy. Future studies should incorporate an extended follow-up period to evaluate the persistence of treatment benefits and identify any potential long-term side effects.

Conclusion

Ligustrazine phosphate tablets combined with cerebroside carnosine can improve the recovery of neurological function, significantly reduce the levels of TNF-α and CRP, and improve the quality of life. However, the sample size in this study is small, and more studies with larger sample size is needed to confirm the results. While the results indicate promising short-term benefits, future studies should explore the long-term efficacy and safety of this combination therapy.