Abstract

Background: Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine produced by macrophages in acute inflammatory processes and plays roles in cell signaling that cause necrosis and apoptosis. This study aimed to show whether there was an effect of Neuroaid (MLC 901) on TNF-α levels in rats with traumatic brain injury (TBI) measured using the Enzyme-linked immunosorbent assay in the peripheral blood.

Methods: A total of 10 Sprague-Dawley rats were divided into two groups, one group was given MLC 901 (n = 5), and the other group was not given MLC 901 (NaCl 0.9%) (n = 5). All groups were treated with brain injury using the modified Marmarou model. The measurements of TNF-α were performed at 30 min and 6 weeks after brain injury.

Results: At 30 min after brain injury, the TNF-α level in the MLC 901 group was higher (3564.8) than the 0.9% NaCl group (3453.6), but it was not statistically significant (P = 0.830). At 6 weeks of treatment, the TNF-α level in the MLC 901 group (2576.6) was higher than the 0.9% NaCl group (1383.4) and statistically significant (P = 0.001). This study showed that the administration of MLC 901 could increase TNF-α levels at 6 weeks after treatment.

Conclusion: MLC 901 increases TNF-α levels in rats with TBI, with a significant rise observed at 6 weeks, suggesting a sustained inflammatory response.

Keywords: MLC 901, Traumatic brain injury, Tumor necrosis factor-alpha

INTRODUCTION

MLC 901 is a traditional medicine in China that contains a simple herbal formulation.[ 13 , 15 ] Several studies suggest that MLC 901 has been shown to prevent cell death and stimulate neurogenesis.[ 8 , 21 ] Treatment of MLC 901 could improve the recovery of memory function in rodents as well as in humans after global ischemia.[ 19 ] In many studies, MLC 901 helps to facilitate the restoration of neural circuits through its antioxidant properties, promotes cell proliferation, and stimulates axonal and dendritic neural circuits after traumatic brain injury (TBI).[ 20 ] Furthermore, the treatment of MLC 901 decreased brain lesions caused by TBI.[ 29 ] Clinical trials of MLC 901 have been successfully carried out for ongoing TBI patients with vascular cognitive impairment.[ 25 ]

TBI is one of the leading causes of death and disability in the world; approximately 1.7 million people have TBI in the United States.[ 11 ] CDC also reports that approximately 5.3 million people in America have side effects of TBI[ 27 ], and this condition is most often faced by neurosurgeons related to advances in science and technology.[ 3 , 5 , 10 ] The clinical features of TBI were prolonged coma, headache, nausea, aphasia, seizures, amnesia, and behavioral disorders.[ 17 ] That condition will lead to brain pathology, impaired normal brain function, and death.[ 14 ] TBI causes early activation of resident microglial and recruitment of peripheral neutrophils.[ 23 ]

Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine produced by macrophages/monocytes during acute inflammation; it is responsible for a wide variety of signaling events in cells that cause necrosis or apoptosis.[ 6 , 9 ] The synthesis and secretion of TNF-α is regulated by TNF-α converting enzyme, a proteinase that is responsible for the breakdown of TNF-α on the membrane surface. Tumor necrosis factor-alpha (TNF-α) exerts a proinflammatory role in the central nervous system (CNS). TNF-α is the main mediator of the inflammatory process that causes a series of cellular cascades in nerve cell death. On the other hand, TNF-α has function as a neuroprotector in certain neurological conditions.[ 24 ]

MATERIALS AND METHODS

This study observed the relationship between administration of MLC 901 with TNF-α in serum and brain tissue after TBI, then compared the responses between two groups: the group that was given MLC 901 and the group that was not given MLC 901.

Animals

Ten Sprague-Dawley rats (aged 10–12 weeks, weight 280– 300 g) received standard food (Comfeed AD-2) and water until research occurs. The rats were divided into two groups: (1) brain injury with NeuroAid administration (MLC 901) and (2) brain injury without NeuroAid administration (MLC 901). This study was approved by the Ethics Commission of the Faculty of Medicine, Hasanuddin University. The surgical procedure was carried out aseptically.

Treatment of brain injury and sampling

Ten Sprague-Dawley rats that fulfill research criteria were randomly divided into two groups: the Control group (group that was not given MLC901) and the treatment group (group that was given MLC901). The treatment group was given ketamine for anesthesia, then performed brain injury by craniotomy with a coronal incision. After the dura mater was seen, the rats were given 20 g of load dropped from 20 cm of height using the modified Marmarou model. The modified Marmarou model is one of the simple brain injury techniques in experimental animals that results in a diffuse clinical situation of brain injury, which is often caused by motor vehicle accidents or falls in humans. The advantage of this model is that reduces the possibility of death in experimental animals, thereby minimizing research costs. Moreover, this model can be considered as a pharmacological intervention for the therapy of brain injury.[ 7 , 16 ] The treatment group received oral administration of MLC 901 with a concentration of 10 mg/mL in drinking water until the experiment was stopped at 6 weeks, 30 min after the brain injury process.[ 20 ]

Measurement of TNF-α levels

Serum levels of TNF-α were an important marker of the neuroinflammatory process, which is measured in peripheral blood using ELISA examination. Serum proteins of TNF-α levels were measured using a sandwich-type ELISA kit that is available on the market. The enzyme-linked immunosorbent assay (ELISA) works to determine the level of target gene protein in serum/plasma. ELISA is one of the biochemical techniques that is mainly used in immunology to detect the presence of an antibody or antigen in a sample. ELISA has been used as a diagnostic tool in the medical field, plant pathology, and also in various industrial fields.

Statistical analysis

The data were processed and analyzed by computer using Excel 2013 and the Statistical Package for the Social Sciences version 22. Expression of TNF-α will be displayed in mean (standard deviation) with confidence intervals (95% CI). A bivariate analysis was performed using the Kruskal-Wallis test and the independent t-test to compare differences between groups. Correlation test was carried out between the dependent variable and added with repeated ANOVA measurement to test the hypothesis and see the effect of intervention. One-way analysis of variance test was used to see the differences between groups, both the positive control group and the treatment group. To see the difference in mean values and probability values, the least significant difference test is used.

Equipment resources

The study was conducted on male Sprague-Dawley rats, body weight 300 g, 3 months old, and placed in a cage with controlled temperature and humidity (23 ± 1°C). The equipment used was anesthetic drugs, microsurgical tools for craniectomy, and iron rods with a weight of 20 g and a diameter of 4 mm, along with guide tubes using the modified Marmarou model.

RESULTS

Effect of MLC 901 on TNF-α levels in rats with brain injury

Before the procedure, TNF-α level in the treatment group (MLC 901 group) was 670.0, and the control group (0.9% NaCl group) was 623.0. At 30 min after brain injury, TNF-α level in the treatment group (MLC 901 group) was higher (3564.8) than the control group (0.9% NaCl group) (3453.6), but it was not statistically significant (P = 0.830), so both groups were at the same baseline. At 6 weeks after treatment, TNF-α level in the MLC 901 group (2576.6) was higher than the 0.9% NaCl group (1383.4) and statistically significant (P = 0.001). This study showed that administration of MLC 901 could increase TNF-α levels at 6 weeks after treatment. The results of statistical tests are shown in Table 1 and Figure 1 .

Table 1:

Independent t-test on TNF-alpha levels in rats with brain injury.

Figure 1:

Tumor necrosis factor-alpha levels with and without administration of MLC 901 in experimental rats based on the treatment time. TNF: Tumor necrosis factor, MLC: Molea

DISCUSSION

TBI is a complex condition involving primary and secondary brain injury. Combined therapy could be a better treatment strategy, using formulations that contain more than one active ingredient. Traditional Chinese medicine (TCM) has been recommended for centuries to treat a wide variety of medical conditions. TCM, which consists of several extracts of herbal medicine, has received attention in the medical field. NeuroAid has emerged as a promising treatment to support neurological recovery. Several clinical trials and reports have established its safety profile.[ 24 ] Research by Tsai et al. found that NeuroAid had a positive effect in reducing brain contusion in rats with brain injury.[ 26 ] Contusions due to TBI were associated with neurological motor deficits, brain apoptosis, and activated microglia.[ 12 ]

TNF-α is one of the inflammatory mediators that play roles in acute and advanced (chronic) phases of brain injury. As a major inflammatory pathway, TNF-α is known to induce cell proliferation and apoptosis. In the acute phase, TNF-α is neurodestructive, but in chronic phase TNF-α is neuroprotective. Research on rats that were given TNF-α inhibitor on 1^st and 12 h post-brain injury showed an increased in cognitive performance, decreased apoptosis, and decreased astrogliosis after 1 week. This study showed that there was a significant difference in the mean of TNF-α expression after MLC 901 administration compared to MLC 901 administration at 6 weeks after brain injury. This result explains that the role of TNF-α in the chronic phase of brain injury is very important as a neuroprotective, thereby giving MLC 901 indirectly in the chronic phase of brain injury is very useful for clinical improvement.[ 1 ] Experimental study in patients with brain injury showed that TNF-α has a role in mediating neuronal cell death in the acute phase and also promotes nerve cell repair and neuroplasticity in the chronic phase with different effect according to activation of subtypes receptor of TNF-α that can affect the outcome in acute phase or chronic phase after brain injury.[ 28 ]

The results of this study were different from the study by Tsai et al. in 2015, rats with brain injury given MLC 901 after 4 days were able to reduce the synthesis of microglia that play roles in secretion of TNF-α; then, it will reduce neurodestructive processes.[ 26 ] However, previous research has shown that TNF-α has a beneficial role in the chronic phase of post-brain injury. TNF-α mediates neurorepair and neuroplasticity processes in the chronic phase. Studies show that TNF-α can participate in peripheral nerve repair in rats, blood vessel repair in humans, and wound healing in rats. TNF-α has been shown to increase spontaneous synaptic flow in hippocampal neurons in vitro, suggesting a role for TNF-α in mediating in vivo synaptic plasticity. TNF-α can also enhance the recovery process by increasing the expression of anti-inflammatory cytokines, such as Interleukin-10 and transforming growth factor-α, and these cytokines have the potential role in chronic CNS repair mechanisms by triggering proliferative responses, astrocytes after injury.[ 22 ]

Inhibition of TNF-α in the early phases of brain injury can be beneficial, but can be detrimental in chronic phases. The decrease of TNF levels can be maximized by pharmacological agents that have an inhibitory effect in the acute phase of brain injury.[ 4 ] Neuroprotective roles of TNF-α were observed in brain injury in rats after 4 days, indicating that there is a temporary repair process in the locomotor performance of rats.[ 2 ] These results are similar to the research of Oshima et al. (2009), TNF-α is involved in the recovery process, especially in functional and/or neuroanatomical plasticity after 14 days of brain injury in rats.[ 18 ] The rate of sprouting axons appears to correlate with functional recovery in this study. Previously has been shown that the axons grows from corticorubral tract after ischemic brain injury were enhanced by blockage of Nogo receptor, which is a key receptor for inhibiting axon growth in the CNS.[ 18 ]

NeuroAIDS (MLC 901) derived from TCM has been shown to have neuroprotective and neurorestorative properties in models of preclinical stroke, global cerebral ischemia, and TBI. NeuroAid (MLC 901) contains 9 herbal components, namely Radix Astragali, Radix Salvia Miltiorrhizae, Radix Paeoniae Rubra, Rhizoma Chuanxiong, Radix Angelicae Sinesis, Carthamus Tinctorius, Prunus Persica, Radix Polygalae dan Rhizoma Acori Tatarinowii.[ 25 ]

A study conducted by Rosyidi et al., (2020) said that the administration of MLC 901 can increase the neurogenesis process, which is marked by an increase in the expression of synaptophysin levels.[ 21 ] These results indicate that the administration of NeuroAid (MLC 901) in TBI has well responds to the enhancement of the neurogenesis process.[ 21 ] NeuroAid (MLC 901) has neuroprotective and neurorestorative effects, leading to enhanced recovery of cognitive function in rats who suffered TBI. This is a great basis for exploring the therapeutic effect of NeuroAid (MLC 901) to improve recovery in patients with TBI.[ 20 ]

CONCLUSION

From the results and descriptions above, it can be concluded that the administration of MLC 901 can increase TNF-α levels in the chronic phase in the rat model with brain injury.

Ethical approval:

This study was approved by the ethics commission of The Faculty of Medicine, Hasanuddin University, with license number: 771/UN 4.6.4.5.31/PP36/2020.

Declaration of patient consent:

Patient’s consent not required as there are no patients in this study.

Financial support and sponsorship:

Nil.

Conflicts of interest:

There are no conflicts of interest.

Use of artificial intelligence (AI)-assisted technology for manuscript preparation:

The authors confirms that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Disclaimer

The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Journal or its management. The information contained in this article should not be considered to be medical advice; patients should consult their own physicians for advice as to their specific medical needs.

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