Clinical efficacy of combined use of Xuebijing and mild hypothermia therapy for the treatment of severe traumatic brain injury, and its effect on patient mortality and complications

Purpose: To investigate the clinical efficacy of combined use of Xuebijing and mild hypothermia therapy in severe traumatic brain injury (TBI), and its effect on mortality and incidence of complications. Methods: Eighty-six severe TBI patients admitted to The Fourth People's Hospital of Chongqing were selected. Patients were randomly and equally assigned to control group (COG) who received conventional treatment, and study group (EXG) given a combination of Xuebijing and mild hypothermia therapy. Relevant clinical indicators and therapeutic effects were compared. Results: Post-treatment levels of inflammatory indices, including procalcitonin (PCT), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly reduced, with lower levels in EXG (p < 0.05). Between-group differences were seen in human leukocyte antigen-DR isotype (HLA-DR), CD3 + , CD4 + , CD8 + , and C3 indicators (p < 0.05). EXG had significantly lower protein expression levels of neuron-specific enolase (NSE) and S100 beta than COG (p < 0.05). After treatment, plasma protein levels of coagulation indices, viz, prothrombin time (PT) and activated partial thromboplastin time (APTT) were lower than pre-treatment levels, with even much lower levels in EXG (p < 0.05). There were reduced incidence of lung infection, acute gastrointestinal injury, acute respiratory distress syndrome (ARDS) and hypernatremia (p < 0.05). More patients had better recovery in EXG (p < 0.05) than in control group. Conclusion: The combined therapy improves the prognosis of severe TBI, lowers the levels of inflammatory factors, ameliorates plasma coagulation, and enhances immunity with a high degree of safety. However, further clinical trials should be undertaken prior to application in clinical practice.


INTRODUCTION
Statistics have revealed that the incidence of severe traumatic brain injury (TBI), which is second only to that of limb fractures, accounts for 15 -20 % of injuries in all parts of the body [1,2]. In recent years, with increased incidence of events such as traffic accidents, falls, and trauma, severe TBI has become an emergency and a critical illness in neurosurgery, and a source of increased morbidity and mortality [3,4]. At present, the clinical treatment of TBI patients is mostly limited to arresting bleeding, lowering intracranial pressure, protecting neurological function, and preventing secondary organ injury. However, these strategies produce unsatisfactory clinical efficacy. Currently, mild hypothermia treatment is used in neurology to reduce intracranial pressure and protect nerve function in the brain. However, this treatment often induces a complication of coagulation disorder in hypothermia state, resulting in some therapeutic limitations. Clinical studies have found that most cases of severe TBI are accompanied by multiple organ dysfunction involving a variety of pathological mechanisms such as inflammatory response and disturbance in coagulation function [5][6][7][8].
Xuebijing suppresses inflammation in patients through multi-pathway and multi-target mechanisms. It is used mostly for the clinical treatment of acute and critical diseases e.g., sepsis. Therefore, it is likely that combination of Xuebijing and mild hypothermia therapy may result in enhanced clinical treatment efficacy. Currently, not much is known about the clinical effect of this combination treatment on severe TBI, as well as the underlying mechanism. Therefore, this research was aimed to investigate the clinical effect of the combination of Xuebijing and mild hypothermia therapy on severe TBI, as well as the underlying mechanism.

METHODS Patients
A total of 86 patients with severe TBI and admitted to The Fourth People's Hospital of Chongqing (January 2019 -January 2020) were selected for this prospective study. Based on their order of admission, they were assigned to control group (COG) which was given basic treatment + mild hypothermia therapy (n = 43), and study group (EXG) given basic treatment + Xuebijing + mild hypothermia therapy (n = 43). This study got the approval of the ethics committee of The Fourth People's Hospital of Chongqing (approval no. 20181172), and complied with the guidelines of Declaration of Helsinki [9]. Written and signed informed consent was obtained from the patients and/or guardians.

Inclusion criteria
The patients enrolled were those who were meeting the diagnostic criteria in the Guidelines for the Management of Severe TBI (2016 version), patients who were diagnosed with severe TBI after cranial CT examination, and patients with GCS scores of 5 -8 points, with coma time duration more than 6 h.

Exclusion criteria
Patients in the following categories were excluded: Those with TBI complicated with severe organ dysfunction or acute infectious diseases, patients with other cardiovascular and cerebrovascular diseases, and those with previous limb motor dysfunction. Moreover, patients with poor treatment compliance, and those who were pregnant or lactating, were excluded.

Treatments
On admission, patients in both groups received conventional treatments according to their conditions. The conventional treatments comprised surgery, reduction of intracranial pressure using dehydration therapy, nourishment of cranial nerves, oxygen inhalation, use of antibiotics, hyperbaric oxygenation, supportive care, acid suppression, and maintenance of homeostasis. During the treatments, the patients' respiratory tracts were kept clear, and low blood oxygen status was actively corrected [10][11][12][13].
In addition to conventional treatment, patients in EXG received combination of Xuebijing and mild hypothermia therapy. With respect to mild hypothermia therapy, within 24 h of injury, HGT-200 double-blanket mild hypothermia therapeutic instrument was used to cool the body of the patient, with rectal and brain temperatures maintained at 32.0 -35.0 °C and 32.5 -35.0 °C, respectively. At the same time, a hibernation and muscle-relaxing mixture (100 mg of chlorpromazine + 100 mg of promethazine + 400 mg of atracurium + 500 mL of normal saline) was administered via intravenous drip at a flow rate of 13 -20 drops/min. The volume and flow rate were adjusted in line with changes in vital signs of patients.
The treatment lasted for 4 -7 days. Slow rewarming of the patients was conducted after cessation of mild hypothermia therapy, with temperature increased at the rate of 1 °C every 4 h, and the administration of hibernation mixture was stopped gradually over a period of 24 h. Xuebijing (50 mL; Tianjin Chase Sun Pharmaceutical Co. Ltd., specification: 10 mL; NMPA approval no. Z20040033) was administered with intravenous micro-dosage pump thrice daily continuously for 5 -7 days. The other treatments were same as in COG [14,15].

Levels of inflammatory factors
Before and after treatment, the serum PCT levels in both groups were determined with fluorescence enzyme immunoassay, while the TNF-α and IL-6 levels were determined with enzyme-linked immunosorbent assay (ELISA).

Serum protein levels of neuron-specific enolase (NSE) and S100 beta
The protein levels of NSE and S100 beta in patients of both groups were determined with ELISA before and after treatment.

Plasma coagulation
Plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured in patients in both groups before and after treatment, using wet chemical method.

Complications
Complications during the treatments were compared between the two groups. These complications comprised lung infection, acute gastrointestinal injury, ARDS, disseminated intravascular coagulation (DIC) and hypernatremia.

Treatment outcomes and mortality
All patients were followed up for six months, and the Glasgow Outcome Scale was used to assess the effect of treatment in each group. Patient's status was rated as good recovery (patient had minor defects but was able to live normally), moderate disability (patient was disabled, but was able to work under protection and carry out basic activities of daily living), severe disability (patient was conscious but unable to take care of themselves without help), or persistent vegetative state. Cases of death were also recorded.

Statistical analysis
Statistical analysis was done with SPSS 21.0 software, while graphics was done with GraphPad Prism 7 (GraphPad Software, San Diego, USA). Enumeration data are expressed as numbers and percentages (n (%)), while measurement data are expressed as mean ± standard deviation (SD). Enumeration data and measurement data were compared using ꭓ 2 test and t-test, respectively. Differences were considered statistically significant at p < 0.05.

Patients' profile
There were no statistical differences in general profile between the two groups (p > 0.05; Table  1).

Levels of inflammatory factors
After treatment, there were reduced levels of PCT, TNF-α, IL-6 and other indicator inflammatory factors in patients in both groups, with lower levels in EXG than in COG (p < 0.05), as presented in Table 2.

Serum protein levels of NSE and S100 beta
The EXG had lower protein levels of NSE and S100 beta than COG (p < 0.05), as shown in Figure 1.

Plasma coagulation indices (PT and APTT)
As shown in Table 4, after treatment, plasma levels of PT and APTT in both groups were markedly reduced, with lower values in EXG than in COG (p < 0.05).

Incidence of complications
There were lower numbers of patients with lung infection, acute gastrointestinal injury, ARDS and hypernatremia in EXG than in COG (p < 0.05). Although the incidence of DIC in EXG was slightly lower than that in COG (p > 0.05). Details are demonstrated in Table 5.    Figure 1: Serum protein levels of NSE and S100 beta. *P < 0.001, NSE level in EXG after treatment vs NSE level in COG after treatment; **p < 0.001, S100 beta protein level in EXG after treatment vs S100 beta protein level in COG after treatment

Treatment outcomes
No statistical differences were seen in cases of moderate disability, severe disability, persistent vegetative state and death between the two groups (p > 0.05). However, after treatment, more patients had good recovery in EXG than in COG (p < 0.05). Details are presented in Table  6.

DISCUSSION
Due to rapid deterioration of severe traumatic brain injury (TBI), patients often experience ischemia and hypoxia of brain tissue, and varying degrees of inflammatory reactions, leading to complications and secondary lesions [16].   Mild hypothermia therapy is often applied for treating TBI. It relieves TBI-induced pathological damage by alleviating cerebral edema, improving cerebral oxygen metabolism and effectively lowering cerebrovascular endothelial cell metabolism. Moreover, mild hypothermia therapy mitigates Ca 2+ -induced nerve cell damage by reducing Ca 2+ influx, protecting brain tissue, and reducing ischemia-reperfusion injury. It achieves these by inhibiting excessive release of harmful substances such as acetylcholine, reactive oxygen species (ROS) and monoamines, and by suppressing lipid peroxidation [17].

Several studies in China have shown that
Xuebijing injection significantly suppressed lipid peroxidation in cardiac muscle cells, alleviated myocardial injury, and reduced inflammatory reactions by enhancing activity of superoxide dismutase (SOD) [18]. Mild hypothermia therapy in combination with Xuebijing injection has been effective in the treatment of severe diseases. However, not much is known about the effect of the combined treatment on severe TBI. Therefore, 86 severe TBI patients were chosen for this prospective study for the investigation on the clinical efficacy of combination of Xuebijing injection and mild hypothermia therapy on the disease. The results showed that after treatment, there were marked reductions in PCI, TNF-α and IL-6 in both groups, with lower levels in EXG than in COG. These are similar to those reported in a previous study [19]. In addition, after treatment, the plasma coagulation indices: PT and APTT were reduced in both groups, but with lower levels in EXG than in COG. Patients with severe TBI often experience brain tissue edema, aggravated cerebral swelling, rapid rise in intracranial pressure, as well as coagulation disorder (a complication). These changes lead to reduction in plasma fibrinogen level, prolongation of PT and APTT, and aggravation of TBI. In addition, traumatic stimulus and oxidative stress activate the immune system and up-regulate inflammatory factors, leading to a series of inflammatory reactions. Pro-inflammatory factors such as PCT, TNF-α and IL-6 reflect the degree of inflammation in vivo, while PT and APTT are reliable indices of fibrinolytic function. Thus, the combined use of Xuebijing injection and mild hypothermia therapy effectively inhibited inflammatory response and improved coagulation function in severe TBI patients.
Furthermore, this study showed that after treatment, levels of HLA-DR, CD3 + , CD4 + , CD8 + and C3 indicators in patients in both groups differed significantly, implying that the combination treatment improved cellular immune function, enhanced neutralization of endotoxins, reduced secondary brain damage, and prevented infection. The proteins NSE and S100 beta are important serum indicators of the severity of TBI. These parameters reflect the degree of brain tissue damage and the degree of blood-brain barrier (BBB) injury through the permeability of brain capillaries. In this study, EXG had lower protein expression levels of NSE and S100 beta than COG, indicating that the combined treatment inhibited brain tissue damage and protected the BBB, most likely due to reduction of in vivo inflammation.
Cases of lung infection, acute gastrointestinal injury, ARDS and hypernatremia in EXG were lower than those in COG, while the incidence of DIC was comparable in both groups. Moreover, more patients had good recovery in EXG than in COG. These results suggest that the incidence of complications associated with severe TBI may be reduced by combined use of Xuebijing injection and mild hypothermia therapy. The alleviation of inflammatory reactions and improvement of coagulation function reduced the incidence of complications, thereby confirming the effectiveness of the combined treatment.
The condition of patients who were treated with Xuebijing injection in combination with mild hypothermia therapy was better than that of those who received only conventional treatment, possibly due to the mitigating effect of the combined treatment on multiple organ dysfunction syndromes. This implies that Xuebijin and mild hypothermia therapy exerted synergistic effect on TBI by reducing excessive inflammatory response, improving coagulation function, inhibiting release of ROS, and protecting vascular endothelial cells. As a result, the combined treatment enhanced resistance to inflammatory response, cell apoptosis and ischemia-reperfusion injury, thereby decreasing damage to various body functions. Therefore, the combination treatment has a high clinical value in the prognosis of severe TBI in patients.

Limitations of the study
The recommended clinical dose of Xuebijing injection for patients with systemic inflammatory response syndrome is 50 mL twice a day, or 50 mL three times a day in severe cases; while the dose for patients with multiple organ dysfunction syndromes is 100 mL twice a day, or 100 mL three times a day in severe cases. However, in this study, there were no control groups given various doses via different administration methods. Therefore, there is need for large-scale prospective studies to determine the optimum dose and administration regimen for the combination treatment. Moreover, since mild hypothermia therapy is not suitable for patients with hemorrhagic shock or severe cardiopulmonary diseases, or those over 70 years, the contraindications and indications of patients should be considered before using the combination regimen.

CONCLUSION
The combination of Xuebijing injection and mild hypothermia therapy improves the prognosis of severe TBI in patients, lowers the levels of inflammatory factors, improves coagulation function, and enhances immunity. However, further clinical trials of the combined therapy on a larger scale is recommended prior to its introduction into clinical practice.

Acknowledgements
None provided.

Funding
None provided.

Ethical approval
None provided.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.