key: cord-0736017-19756j6j
authors: Luo, Zhijian; Chen, Wei; Xiang, Mingqing; Wang, Hua; Xiao, Wei; Xu, Cheng; Li, Yunkui; Min, Jie; Tu, Qiang
title: The preventive effect of Xuebijing injection against Cytokine Storm for severe patients with COVID-19: a prospective randomized controlled trial
date: 2021-01-30
journal: Eur J Integr Med
DOI: 10.1016/j.eujim.2021.101305
sha: b20fe7e99c8f4b3306bde6e30e5447a683c53588
doc_id: 736017
cord_uid: 19756j6j

INTRODUCTION: COVID-19 is severely affecting countries globally and mortality is high. Xuebijing (XBJ) injection is widely used in the treatment of severe pneumonia and sepsis in China due to its anti-inflammatory effect and immunoregulation. This study investigated whether Xuebijing injection can prevent the cytokine storm and reduce the mortality from severe COVID-19. METHODS: This was a randomized, double-blinded trial in which 60 eligible patients were recruited from the First people's Hospital of Jingzhou from February 16 to March 25 in 2020. A total of 57 completing the trial, 3 dropped out. Treatment group received routine medication plus Xuebijing while control group received routine plus saline. RESULTS: The secretion of interleukin-6(IL-6), interleukin-8(IL-8) and tumor necrosis factor-α(TNF-α) was suppressed significantly (P<0.05) by Xuebijing. After 14 days treatment, lymphocyte levels in Xuebijing group was substantially higher than control, C-reactive protein (CRP) level in Xuebijing group was remarkably lower. The 28-day mortality was not significantly different between the two group. After 14 days of treatment, there were significant differences in the rate of mechanical ventilation, rate of septic shock, the proportion of patients severely affected who became critically ill type, the duration of improvement of main clinical symptoms (P<0.05) and the length of ICU hospitalization stay (P<0.01) the Xuebijing group.No serious adverse reactions were found for all patients. CONCLUSIONS: This study demonstrates that Xuebijing may suppress the CS in severe COVID-19 patients by regulating the secretion of pro- inflammatory cytokine IL-6, IL-8 and TNF -α. However, Xuebijing did not significantly reduce the 28-day mortality.

In late December 2019, a lethal pneumonia was reported in Wuhan, China caused by a new severe acute respiratory syndrome coronavirus2 (SARS-CoV-2) variant. The novel virus was subsequently named as Corona Virus Disease 2019(COVID -19) by the World Health Organization [1] . The virus spread so rapidly and widely and as of 18 December 2020, a total of 73 575 202 confirmed cases and 1 656 317 deaths had been reported in 222 countries according to the daily report of the World Health Organization (https://www.who.int/emergencies/diseases/novel-coronavirus-2019)

The pandemic continues to spread rapidly and globally. Patients with mild symptoms exhibit a fever and dry cough. The severe cases are more likely to develop dyspnoea, multiple organ dysfunction syndrome (MODS ), irreversible coagulation disorders, uncorrectable metabolic acidosis, septic shock and acute respiratory distress syndrome (ARDS) [2] [3] [4] . Overall mortality for COVID-19 patients is about 4% [3, 5] , then this number reach up to 60% for critically ill patients in the intensive care unit [6] .COVID-19 can be divided into four types according to the severity as follows: mild, moderate, severe and critical illness [7] . Patients with mild disease can become severe cases in a very short time. It has been reported that about 20% of COVID-19 patients may develop into severe cases [5] and severe COVID-19 patients have a high risk of mortality [2] .

Cytokine storm (CS) is very common for severe COVID-19 patients and it means excessive and uncontrolled release of pro-inflammatory cytokines [5] . Inflammatory CS is associated with the progression and development of COVID-19. The level of serum cytokines in COVID-19 patients with ARDS is significantly increased, and it has been reported that the level of serum cytokines is positively correlated with mortality [8] . Accumulating evidence suggests that patients with severe COVID-19 might have a cytokine storm syndrome. A variety of pro-inflammatory cytokines, including interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-α), play a critical role in CS. CS can contribute to the development of ARDS which is the leading cause of death in patients with COVID-19 [9, 10] .

Currently, there is no approved treatment for COVID-19 [11] . Chinese clinical immunologists argue that traditional Chinese medicine and pharmacology, attributing to inhibition of excessive inflammation and immunoregulation, are required to be used in the treatment of COVID-19 [5] . Growing evidence shows some traditional Chinese herbs and medicines may have an immunosuppressive effect [12] . Li et al reported that a Chinese patent medicine named Shuanghuanglian oral liquid combined with western medicine successfully cured three confirmed patients with COVID-19 in Wuhan [13] . Xuebijing (XBJ) injection is an intravenous herbal preparation made from five traditional Chinese medicines which are composed of Radix Paeoniae Rubra, Angelica Sinensis, Chuanxiong, Honghua (Flos Carthami) and Salvia Miltiorrhiza. Based on the clinical effectiveness and safety on XBJ injection in the treatment of infectious diseases such as severe pneumonia and sepsis [14] [15] [16] , it was recommended as one of the treatment options for COVID-19 patients in the diagnosis and treatment guidelines which was issued by National Health Commission of the People`s Republic of China [12] . Network pharmacology suggests that Xuebijing may alleviate COVID-19 patients` inflammatory reaction through regulating some cytokines such as TNF [17] . So we conducted a prospective, randomized, double-blinded trial in order to investigate whether Xuebijing injection can suppress the cytokine storm and reduce the mortality of severe COVID-19 patients.

This study was a single-center, prospective, randomized, double-blinded trial. 2) oxygen saturation less than 93%.

3) arterial partial pressure of oxygen (PaO2) / concentration of oxygen (FiO2) ≤300mmHg. pulmonary imaging showed that patients with obvious progress of lesions more than 50% in 24-48 hours. 6. Glucocorticoids were used during hospitalization.

1. Poor medical compliance.

A serious adverse event such as allergic shock occurred 3. Using of drugs or treatments that may affect the outcome. Antivirus drug such as lopinavir and ritonavir Tablets and hydroxychloroquine, blood purification treatment.

4. If the patient die within one week after enrollment, the patient is classified as drop out case.

Of the 82 COVID-19 patients from Jingzhou, Hubei province, 60 met the eligibility criteria and agreed to take part. When the patients themselves or their close relatives signed the informed consents, these patients were assigned randomly to the XBJ or control group with a 1:1 ratio by a central randomization system. XBJ group received routine medication plus XBJ injection, and 50 ml XBJ injection was diluted with 100 ml normal saline to 150 ml, every 12 h for 60 min. The manufacturer of XBJ injection is Tianjin Chase Sun Pharmaceutical Co., Ltd., Tianjin, China (lot number Z20040033). Control group patients received the same routine medication plus 150ml saline. The routine medication included nutritional support, oxygen therapy, antiviral therapy with interferon-α inhalation, antibiotic agents, noninvasive and invasive ventilation if necessary. Both groups received the routine medication for 14 days starting within 24 hours of enrollment.

Randomization was generated centrally by a computerized random-number generator which drew up an allocation schedule for XBJ group and control group with a ratio of 1:1. Each group would then receive the corresponding treatment regimen.

Firstly the drug administrators received group information based on the above mentioned random number, then they dispensed the drug to the nurses, who made the mixture of drugs in a separate room. Photophobic brown color infusion bags and infusion set was prepared to ensure the same appearance.

The drug administrators and dispensing nurses were not involved in the data collection and analysis. Drug administrators and the investigators logged on the central randomization system using independent authority and accounts. The randomization system concealed group information for all the patient.

The primary outcome was the level of peripheral blood lymphocyte and IL-6 on the 1st, 7th, 14th day of the enrollment, The secondary outcome measures were as follows:

(1) 28-day follow-up mortality, the rate of mechanical ventilation, septic shock and conversion to the moderate type during 14 days of treatment. (2) Peripheral blood leukocytes, C-reactive protein (CRP), IL-8 and TNF-α on the 1st, 7th, 14th day of the enrollment. Sample collection was carried out before breakfast on the specific day.

Serum samples were assayed with IL-6, IL-8, and TNF-α enzyme-linked immunosorbent assay (ELISA) kit respectively (Abcam; Cambridge, MA, USA). The 

The sample size was calculated based on the hypothesis that the effects of XBJ injection on severe COVID-19 patients would be similar to a previous study on severe pneumonia [18] . The sample size was estimated to be 60. The statistical software is SPSS version 20.0. Significance level is 0.05, two-sided. An unadjusted χ2 test was used for proportions for 28-day mortality. Two independent samples t-test was the analysis method for normal distribution data. P<0.05 means statistical significance. Chi square test was used to analyze the categorical variables expressed as a percentage. Repeated measures analysis of variance was used to compare multiple groups of data. Descriptive statistics were used for demographics, baseline characteristics, and safety variables. All analyses were performed according to the Intention-To-Treat principle.

A total of 82 COVID-19 patients were screened for eligibility and 60 met eligibility criteria. Using the central randomization system, participants were randomly divided into either XBJ or control group, 30 for each. 57 of them completed the trial. 1 case in XBJ group and 2 cases in control group died within one week and were classified as drop out cases ( Figure. 1). Baseline characteristics of patients in both groups, such as gender, age, systolic blood pressure, respiratory rate, heart rate, comorbidities, use of antibiotics are summarized in Table 1 .

As shown in Table2, the incidence of ARDS and mechanical ventilation in XBJ group was lower than that for the XBJ group after 14 days of treatment (P<0.05).

Meanwhile, the septic shock rate of XBJ group was significantly lower than that of the control group within 14 days (P<0.05). 28-day mortality was not significantly different between the two groups (1 [3.45%] died in the XBJ group vs 7 (25%) in the control group, although numerically higher in the control group; (P>0.05), (Table2).

The results in Table3 show that the proportion of patients in the XBJ group who became critically ill was lower than that of control group after 14 days treatment (P<0.05) and the XBJ group were more or less likely to develop moderate illness compared with the control group (P<0.05). As shown in Table 4 , duration of cardinal symptoms such as fever, cough, shortness of breath, fatigue in the XBJ group was shorter than that for the control group (P<0.05). XBJ group also had a remarkably shorter duration in hospital in ICU in comparison with the control group (P<0.01).

As shown in Figure 2A , the two groups have similar peripheral blood leukocyte count on the 1st day (7.37 ± 4.42 in XBJ group, and 5.80 ± 2.56 in control group; P >0.05). There was no significant difference in peripheral blood leucocyte count on the 7 th and the 14 th day in the Xuebijing group compared with the control group ( Figure 2A ). No significant difference was observed in lymphocyte count between the two groups on the 7 th day (P>0.05), whereas the figure on the 14 th day of enrollment was 1.34±0.56 for XBJ group, and 1.02±0.36 for control, with the difference being significant (P<0.05 Figure 2B ). The level of CRP for XBJ group decreased and that of control group slightly increased on the 7 th day with no significant difference between the two groups (P>0.05), then the level of CRP on the 14th day of enrollment was 6.23±11.86 for XBJ group, and 1.02±0.36 for control group (P<0.01 Figure 2C ).

No significant differences were observed in the level of pro-inflammatory cytokines IL-6, IL-8 and TNF-α between the two groups on the 1st day (P>0.05), then the level of IL-6 on the 7 th day of enrollment was 0.042±0.016 for XBJ group, and 0.057±0.014 for control group (P<0.05 Figure 2D ). In addition, the level of cytokines IL-6 declined on the 14 th day of enrollment, 0.03±0.013, 0.04±0.014 for XBJ group and control one respectively (P<0.05 Figure 2D) . Similarly, the level of IL-8 decreased on both the 7 th and 14 th day, with the difference being significant between the two groups (0.28±0.14 vs 0.32±0.15;0.24±0.13 vs 0.30±0.14 P<0.05 respectively; Figure 2E ). Figure 2F ).

There were 5 cases abnormal liver dysfunction, 3 cases renal dysfunction, 2cases rash in the XBJ group and 3 cases abnormal liver dysfunction, 4 cases renal dysfunction, 1 cases rash in the control group. Thus there were no obvious difference between the two groups(P>0.05; Table5). No anaphylactic shock occurred in both groups.

Globally the number of patients with COVID-19 is rapidly increasing and the number of deaths is rising. Some of the patients with mild illness develop rapidly into severe and critical illness, and die within in a short period of time. Approximately, 70% of COVID-19 patients suffered from ARDS and needed mechanical ventilation in ICU. Overall mortality of patients with COVID-19 is about 4% [3, 5] , while 28-day mortality for severe and critical patients reaches up to 60% [6] . With reference to other research designs, lymphocyte count and IL-6 level were taken as the primary outcomes of this study [19] . The results showed that XBJ injection did not significantly reduce the mortality rate, which may be related to the small sample size.

Although 28-day mortality rate was not statistically significant, after XBJ injection intervention, the rates of ARDS and mechanical ventilation in patients with COVID-19 were obviously decreased, further XBJ injection also inhibited septic shock to a large extent. Moreover, the main inflammatory markers such as lymphocyte count and IL-6 level were evidently improved, which indicated that XBJ is effective for severe patients with COVID-19. According to Diagnosis and Treatment Protocol for COVID-19 released by National Health Commission of the People`s Republic of China (Version 6), COVID-19 can be divided into four types as follows: mild, moderate, severe and critical illness [7] . Compared with the control group, the number of severe patients deteriorating to critical illness in the XBJ group was less, and more severe patients developed into more moderate cases. The XBJ group also had a marked shorter hospital stay in ICU. This suggests that XBJ injection may be effective in preventing the progression of COVID-19 and improve the prognosis of patients.

According to relevant literature, the peripheral blood leukocytes of patients with COVID-19 are usually normal, the level of CRP in the peripheral blood of patients with COVID-19 increases significantly, whereas the lymphocyte count decrease greatly. Studies have shown that the severity of COVID-19 is positively correlated with CRP level and negatively correlate with lymphocyte level. Lymphopenia is a common feature and might be a critical factor associated with the mortality in the patients with COVID-19 [20, 21] . Flow cytometry shows that peripheral blood T lymphocyte decreases and be activated. Lymphocytopenia is a marked feature for severe and critical COVID-19 patients. More than 80% of critical patients have lymphopenia. The degree of lymphocyte decreased reflects the severity of infection of SARS-CoV-2. There is a progressive decrease in lymphocytes in patients who died of COVID-19 [21] . Total lymphocyte count is an independent risk factor associated with disease progression in patients with COVID-19 [22, 23] . Lymphocytopenia may be closely related to prognosis. Excessive inflammatory response caused by SARS-CoV-2 is regarded as a major cause of death in COVID-19 patients. This is related to lymphocytopenia and infiltration of monocytes into lung and other important organs [24] , so that lymphopenia may be increasing the risk of death in those with COVID-19 infection. It seems that if the level of peripheral blood lymphocytes was restored, the prognosis of COVID-19 patients would be improved.

So the results imply that XBJ injection have an inhibitory effect on the lymphocytopenia, indicating that XBJ injection might have the effect of regulating cellular immune function for severe COVID-19 patients.

There is also a positive correlation between CRP level and the risk of organ failure in critically ill COVID-19 patients [25] . A majority of the patients with severe COVID-19 in ICU have sustained high level of CRP [5] , and the level of CRP can predict the severity of COVID-19 [23] . Compared with conventional treatment, the expression of CRP for patients with severe COVID-19 was significantly inhibited by XBJ injection on the 14th day of enrollment, suggesting that the inflammatory response was under control.

TNF is considered to be the crucial cytokine in acute viral diseases. TNF is expressed by a variety of immune cells and it plays an important role in CS [9, 27] Tumor necrosis factor alpha (TNF-α) plays a key role in acute inflammation which was induced by oxidative stress and synthetic reaction. TNF -α is produced by macrophages, monocytes, B cells and other tissues. Activation of TNF-α also leads to the production of IL-1 and IL-6. A large number of TNF -α were found in the tissues and plasma of patients with COVID-19. Anti TNF -α antibodies can reduce the early inflammatory response of COVID-19, so that it is a promising therapeutic option for COVID-19 patients [28] . Severe COVID-19 patients have immune dysfunction which was caused by CS. The decrease of lymphocytes may be due to the destruction of CS [5] . SARS-CoV-2 targeting mainly on lymphocyte may cause T cell consumption which is conducive to immune dysfunction. Qin C et al. proposed that serum IL-6, IL-8 and TNF-α levels are significantly higher in patients with severe COVID-19 than those of individuals with mild disease [29] . The CS is closely related to COVID-19 severity [29, 30] ,and it is thought to be a primary cause of death from COVID-19 [24, 31] IL-6 plays a key role in CS. IL-6 is secreted by endothelial cells under the stimulation of hypoxia and inflammatory cytokines such as bacterial lipopolysaccharide and TNF-α. High levels of IL-6 can activate vascular endothelial cells as well as coagulation pathway, and eventually leads to sepsis and MODS [32, 33] . For severe COVID-19 patients, CS is closely related to death. IL-6 is regarded as a new target for COVID-19. If the signaling pathway of IL-6 is blocked, CS can be effectively inhibited. Then, the systemic inflammatory response can be prevented, contributing to the reduce of mortality [10, 33, 34] . In the present study, compared with conventional treatment, the expression of IL-6, IL-8 and TNF-a was substantially down regulated in patients with severe COVID-19 after the treatment with XBJ injection for two weeks, meanwhile the level of CRP significantly declined.Therefore, the present study suggests that CS is suppressed and systemic inflammatory response is under the control in patients with severe COVID-19.

The balance of pro-inflammatory and anti-inflammatory mechanisms is critical to maintain the immune homeostasis of the lung. Missing or abnormal for one or more of these regulatory mechanisms may lead to a CS. SARS-CoV-2 infection induces an excessive immune response and the high level of pro-inflammatory cytokines such as IL-6, IL-8 and TNF -α in patients with severe infection. The secretion of these proinflammatory cytokines leads to CS. And then excessive cytokine release is considered to be the determinant of ARDS [9, 27] . Prevention and mitigation of cytokine storm is considered as the key to save severe COVID-19 patients [31] . T cells play a critical role in antiviral immunity. Recent studies have demonstrated that SARS-CoV-2 viruses can induce reduction and functional exhaustion of T cell in COVID-19 patients, and it may be due to high levels of serum IL-6. TNF -α and IL-6 having a negative regulatory effect on T cell survival or proliferation [35] . After two weeks of intervention with XBJ injection, the levels of IL-6, IL-8 and TNF -α in patients with severe COVID-19 were remarkably reduced, and it indicated that XBJ injection may suppress the CS by inhibiting the reduction of T cells. (Figure3) XBJ injection is a traditional Chinese Medicines which are composed of Radix paeoniae rubra, Angelica sinensis, Chuanxiong, Honghua (Flos Carthami) and Salvia miltiorrhiza, etc [36] .The main ingredients of XBJ injection include amino acid, phenolic acid, flavonoid glycoside, elysine and phthalic acid ester. XBJ exerts effects on about 10 kinds of sepsis / inflammation pathways, and its 21 main active components participate in the regulation of 550 targets. Then XBJ injection possesses biological activity of promoting blood circulation and removing blood stasis and detoxifcation, therefore it is used in the treatment of sepsis with approval [15, 37] .

Basic research and clinical trials have confirmed that XBJ injection could suppress the secretion of proinflammatory cytokine IL-6, IL-8 and TNF-α, and alleviate the injury of severe pneumonia and sepsis [14, 15, [37] [38] [39] . Our result suggests that XBJ injection can also down regulate the expression of pro-inflammatory cytokines IL-6, IL-8 and TNF-α in severe COVID-19 patients.

This study was subject to some limitations. Firstly, XBJ injection is only available in China and treatment setting and protocol is different in other countries. Secondly, sample size of this study was relatively small and further studies including a larger number of patients with severe COVID-19 may be required to validate these findings in future.

In summary, It is suggested that XBJ injection may prevent severe COVID-19 patients against CS by regulating the secretion of pro-inflammatory cytokine IL-6, IL-8 and TNF -α. NMV, noninvasive mechanical ventilation (including high flow supply and face mask). 

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Acknowledgements: Dr. Tang-Meng Guo, MD is acknowledged for his careful review of this manuscript, and precious proposal for improvement.

There were no any commercial involvement in the conduct of the study.None of author is a member of the editorial board.