a b s t r a c t

Purpose: To assess the efficacy of Shenfu injection (SFI) for enhancing cellular immunity and improving the clin- ical outcomes of patients with septic shock.

Methods: Patients with sepsis were randomly assigned to receive either SFI at a dose of 100 mL every 24 hours for 7 consecutive days or a placebo in addition to conventional therapy. The immunologic parameters were collected on days 1, 3, and 7 after the above treatments, and the clinical outcomes were updated for 28 days.

Results: Of these160 patients, 3 were excluded from the analysis due to protocol violation and withdrawal of con- sent; thus, 157 completed the study (78 in the SFI group and 79 in the Placebo group). We found that SFI in- creased both CD4⁺ and CD8⁺ T cells in peripheral blood and up-regulated HLA-DR expression in monocytes (P b .05). Furthermore, SFI was also found to restore ex vivo monocytic tumor necrosis factor ? and interleukin 6 Proinflammatory cytokine release in response to the endotoxin (P b .05). Importantly, the SFI group showed better clinical outcomes than did the placebo group in terms of the duration of vasopressor use (P = .008), Acute Physiology and Chronic Health Evaluation II score (P = .034), Marshall score (P = .01), and length of in- tensive care unit stay (10.5 +- 3.2 vs 12.2 +- 2.8 days; P = .012). However, the 28-day mortality rate was not sig- nificantly different between the SFI (20.5%; 16/78) and placebo groups (27.8%; -22/79; P = .28).

Conclusion: These findings suggest that SFI can enhance the cellular immunity of patients with septic shock and

could be a promising adjunctive treatment for patients with septic shock.

(C) 2016

1. Introduction

Despite the recent advances in medicine, sepsis is still one of the lead- ing causes of death among patients in the intensive care unit (ICU) world- wide [1-4]; moreover, a continued increase in incidence has been observed over recent years [5]. Although sepsis or septic shock is tradi- tionally viewed as an excessive systemic inflammatory reaction to inva- sive microbial pathogens, pharmacologic suppression of the innate immune response has proven unsuccessful [6-9]. An important reason may be that most patients with septic shock survive after the initial pro- inflammatory hit, but die in the subsequent immunosuppressed state

? Financial support: None.

?? Funding: The current study was supported by the Seed Foundation of the Shanghai

University of Medicine & Health Sciences (key project) (Grant No.: HMSF-16-21-026).

? Conflict of interest: The authors declare no conflict of interest.

* Correspondence author at: Zhoupu Hospital Affiliated to Shanghai University of Med- icine & Health Sciences, 1500 Zhouyuan Rd, Shanghai 201318, China. Tel.: +86 21

68135590; fax: +86 21 68135715.

E-mail address: [email protected] (Z. Qiu).

¹ Co-first authors.

due to secondary/opportunistic infections [7,10-16]. Thus, this so-called immunosuppression has been increasingly recognized as the overriding immune dysfunction in patients with septic shock. Therefore, we hypoth- esized that the reversal of sepsis-induced immunosuppression could be a promising adjunctive treatment for those patients.

Shenfu injection (SFI), which originated from Shenfu decoction, a well-known traditional Chinese formulation restoring “Yang” from col- lapse, tonifying “Qi” for relieving desertion, is prepared from ginseng (Panax; family: Araliaceae) and aconite (Radix aconiti lateralis preparata, Aconitum carmichaeli Debx; family: Ranunculaceae). Ginsenosides and aconite alkaloids are the main active ingredients in Shenfu [17]. Ginsenosides are the determinant contributor to the vasodilator benefit of Shenfu, whereas the alkaloids play a vital role in the cardiac electro- physiological effect of Shenfu by blocking ion channels [18]. The SFI has been widely used to treat septic shock in China for more than 2 de- cades; it is frequently used in emergency departments and ICUs in China and has been shown to have some beneficial effects in rescuing these patients [19]. Animal experiments have also confirmed that SFI has ef- fects on scavenging free radicals [21], inhibiting inflammatory media- tors [20,22], suppressing cell apoptosis [18,23], and regulating the host immune response [24,25]. We hypothesized that by enhancing cellular

http://dx.doi.org/10.1016/j.ajem.2016.09.008

0735-6757/(C) 2016

immunity, SFI may help restore immunity, thereby improving outcomes in sepsis. In this randomized control trial, we evaluated the efficacy of SFI in sepsis.

1. Materials and methods

The institutional review board of the Sixth Affiliated Hospital of Wenzhou Medical College approved the study protocol and the partici- pants provided written informed consent. The study was conducted in the 30-bed ICU of a large Tertiary teaching hospital from June 2010 to November 2012.

Patient selection

The inclusion criteria were as follows: age >=18 years and onset of sepsis/septic shock within the previous 24 hours. According to the Third International Consensus Definitions for Sepsis and Septic Shock, sepsis was defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, and septic shock was clinically identified by a Vasopressor requirement to maintain a mean arterial pressure of 65 mm Hg or greater and Serum lactate level greater than 2 mmol/L in the absence of hypovolemia [9].

The exclusion criteria were as follows: known hypersensitivity to SFI, pregnancy, systemic autoimmune disease, hematologic disease (neoplasm, acute leukemia), transplant patients, known HIV infection, and moribundity.

Randomization and blinding methods

Randomization was performed by an independent statistician by generating allocation numbers based on a random number creation sys- tem. The patients were randomized at a ratio of 1:1 to receive either SFI or placebo (0.9% saline) in addition to Standard care. The patients, inves- tigators, and all others involved in conducting the study remained blinded to the treatment assignments for the duration of the study.

Interventions

The SFI was produced by Ya’an Sanjiu Pharmaceutical Co Ltd (Si- chuan, China; batch number: Z20043117). Fifty milliliters of normal sa- line was used to wash the pipeline before and after the administration of SFI. Therefore, the SFI did not interact with other routine medicines. A previous study with a septic rat model showed that a high dose (100 mg/kg) inhibited endotoxin-induced pulmonary inflammation in vivo [26] and ameliorated the mucosal barrier function [27]. Further- more, recent clinical trials have indicated that a dose of 100 mL/d had better clinical outcomes, with special reference to decreasing consump- tion of vasoactive drugs, improving hemodynamics parameters, and shortening ventilator time and ICU stay [28-30]. Thus, the patients in the SFI group received 100 mL SFI in 250 mL of 5% glucose intravenously every 24 hours for 7 consecutive days.

Immunologic parameters

Blood samples were obtained on day 1 as baseline and on days 3 and 7 after randomization for the measurements of T-lymphocyte numbers (including CD4⁺ and CD8⁺ subsets) and monocyte human leukocyte antigen-DR (mHLA-DR) expression. These measurements were per- formed using flow cytometry (BD FACS Aria, BD Biosciences, San Jose, CA) following the manufacturer’s manual. To assess monocytic function, ex vivo endotoxin (lipopolysaccharide [LPS])-induced tumor necrosis factor-? (TNF-?), interleukin (IL) 6, and IL-10 cytokines released from monocytes were analyzed as previously reported [31].

Clinical outcomes

The primary outcomes were the length of ICU stay, the duration of vasopressor use, illness severity, and the degree of organ dysfunction, and the secondary outcome was the rate of death from any cause at 28 days after treatment. The severity of systemic illness was assessed with the Acute physiology and chronic health evaluation II score, with scores ranging from 0 to 71 and higher scores indicating more severe illness [32]. Organ function was assessed weekly with the use of the Marshall score, which ranges from 0 to 4 for each of 6 compo- nents (respiratory, renal, liver, cardiovascular, coagulation, and central nervous system), with higher scores indicating more severe organ dysfunction [33].

Treatment

In addition to the study medication, the patients received standard care from the attending physician according to the Surviving sepsis campaign guidelines, including source control, antibiotics, fluid resusci- tation, vasopressors (noradrenaline, or dopamine), Glycemic control, enteral or parenteral nutrition, and intensive life support (including me- chanical ventilation, Continuous renal replacement therapy, etc). Anti- microbial treatment in both groups followed the recommendations previously described [34]. In brief, anti-infection principles included the following: (1) administration of effective intravenous antimicro- bials within the first hour of recognizing septic shock and sepsis; (2) ini- tial empiric anti-infective therapy using 1 or more drugs that have activity against all likely pathogens (bacterial and/or fungal or viral) and that penetrate in adequate concentrations into the tissues pre- sumed to be the source of sepsis; (3) daily reassessment of the antimi- crobial regimen for potential de-escalation; (4) empiric Combination therapy lasting no more than 3 to 5 days (de-escalation to the most ap- propriate single therapy should be performed as soon as the susceptibil- ity profile is known); and (5) typical therapy duration 7 to 10 days; longer courses may be appropriate for patients who have a slow Clinical response, undrainable foci of infection, bacteremia with Staphylococcus aureus, and some fungal and viral infections or immunologic deficien- cies, including neutropenia.

Safety assessment of SFI

Safety was monitored by observing parameters including changes in symptoms, vital signs, complete blood cell count, and urine routine. To detect any possible side effects of the SFI on the liver and kidney, the serum levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, urea nitrogen, and creatinine were evaluated.

Statistical analysis

To detect an absolute 20% difference in mortality between the SFI group and the placebo group (per previous studies, approximately 40% mortality at 28 days for the placebo group) with an 80% power at a 2-sided P value of .05, 160 patients needed to be included in the trial. All analyses were conducted on a modified intention-to-treat basis. Safety was assessed in the trial population, defined as all subjects who received even a single dose of the study medication. The Kolmogorov-Smirnov test was performed to assess the normal distribu- tion of continuous data. Continuous variables were compared using the unpaired t test or the Wilcoxon rank sum test, and Pearson ?² tests or Fisher exact test was used for categorical variables. For survival analysis, Kaplan-Meier estimates were generated, and between-group differ- ences were assessed using the log-rank test. All analyses were per- formed using SPSS20.0 software (SPSS, Chicago, IL). P values less than

.05 were considered to indicate significant differences.

1. Results
   1. Patients

In the current study, a total of 157 patients, 78 in the SFI group and 79 in the placebo group, underwent follow-up of at least 28 days. Three patients were excluded from the analysis because of protocol vi- olation by 1 patient and withdrawal of consent by 2 patients (Fig. 1).

The 2 groups were well matched at the baseline with respect to de- mographic and clinical characteristics (Table 1). The most common in- fection site was the respiratory tract, followed by the urinary tract, abdomen, blood, and skin or skin structure. In these 2 groups, the inci- dence of positive cultures with gram-negative, gram-positive, or fungal organisms was found to be similar. The isolation of gram-negative or- ganisms was approximately twice as common as the isolation of Gram-positive organisms, and approximately one-third of the cultures were polymicrobial (Table 1).

Immunologic parameters

Compared with the placebo, SFI administration increased signifi- cantly the patients’ CD4⁺ and CD8⁺ T-cell counts, respectively, upon 7-day treatment (Fig. 2A and B; P b .05).

The monocytic function was determined by evaluating the mHLA- DR expression and ex vivo cytokine release in response to the endotox- in. Compared with the baseline, the expression rate of mHLA-DR was significantly elevated in the SFI group on days 3 and 7 (P b .05; Fig. 2C). Furthermore, the LPS-induced monocytic TNF-? and IL-6 re- leases were significantly higher in the subjects treated with SFI on days 3 and 7 (Fig. 2D and E). In contrast, IL-10 release showed a trend toward a lower level in the SFI group (Fig. 2F).

Regarding ex vivo cytokines, the flow cytometry analysis demon- strated that the SFI treatment resulted in an increased frequency of

Table 1

Demographic and clinical characteristics of the patients at baseline

Characteristic	Placebo (n = 79)	SFI (n = 78)	P
Male sex, n (%)	45 (56.9)	43 (55.1)	.817
Age (y)	58.6 +- 17.2	59.3 +- 16.4	.397
Weight (kg)	65.2 +- 15.5	67.3 +- 15.8	.201
APACHE II score	18.3 +- 6.5	18.6 +- 6.8	.389
Marshall score	11.5 +- 4.5	11.2 +- 4.6	.340
Shock, n (%)	61 (77.2)	60 (76.9)	.965
Mechanical ventilation, n (%) Infection site, n (%)a	47 (59.5)	48 (61.5)	.793
Lung	40 (50.6)	37 (47.4)	.689
Urinary tract	17 (21.5)	17 (21.8)	.967
Intra-abdominal	15 (18.9)	14 (17.9)	.867
Blood	7 (8.9)	8 (10.3)	.766
Skin or skin structure Type of organism, n (%)a	5 (6.3)	6 (7.7)	.738
Gram-negative	33 (41.8)	34 (43.6)	.818
Gram-positive	16 (20.3)	17 (21.8)	.813
Fungal	5 (6.3)	4 (5.1)	1.000
Polymicrobial	24 (30.4)	22 (28.2)	.765

The SFI group was treated with SFI; the placebo group was treated with a placebo.

^a Each patient may be counted in more than 1 category.

HLA-DR/CD14 expression in monocytes on day 7, compared with the placebo (Fig. 2C).

Clinical outcomes

As presented in Table 2, the length of ICU stay was significantly shorter (10.5 +- 3.2 days) in the SFI group than in the placebo (12.2 +- 2.8 days; P = .012). The APACHE II and Marshall scores were significantly lower in the SFI group than in the placebo on day 7 after the treatment began (P = .034 and .01, respectively). There was also a

Fig. 1. CONSORT flow diagram.

Fig. 2. Compared with placebo, SFI treatment enhanced the cellular immunity of patients with septic shock. A, CD4⁺ T cells. B, CD8⁺ T cells. C, mHLA-DR expression ratio. D, TNF-?. E, IL-6. F, IL-10. Data were shown as the mean (SD). ?P b .05; ??P b .01 vs day 1 (within-group); ^#P b .05; ^##P b .01 vs placebo.

significant reduction of vasopressor use in the SIF group (2.5 +- 1.5 vs 3.7 +- 1.7 days; P = .008).

Despite a trend, no significant difference was observed in the 28-day mortality between the SFI (20.5%; 16/78) and the placebo group (27.8%; 22/79; relative risk, 0.74; 95% confidence interval, 0.42-1.29; P = .28). A Kaplan-Meier analysis of survival yielded similar results between the 2 groups for cumulative mortality on day 28 (P = .26).

1. Discussion

In addition to clinical parameters investigated, the current study provided a randomized, controlled trial on the immunologic character- istics of sepsis response on patients undergoing SFI. We found that SFI

Table 2

Clinical outcomes of patients with septic shock after treatment

	Placebo (n = 79)	SFI (n = 78)	P
Dead on day 28, n (%)	22 (27.8)	16 (20.5)	.28
ICU stay (d)	12.2 +- 2.8	10.5 +- 3.2	.012

therapy for sepsis was an effective immune-adjuvant measure for re- storing monocyte immunosuppression. Moreover, SFI reduced the du- ration of vasopressor use, the severity of illness, and the degree of organ dysfunction, and it shortened length of ICU stay and tended to de- crease the 28-day mortality.

Sepsis is typically characterized by initial cytokine-mediated hyperinflammation. This hyperinflammatory phase is followed by immu- nosuppression [35,36], which independently contributes to the increased risk of subsequent Nosocomial infections and multiple-organ dysfunction system [37].T cells are often referred to as the “directors” of the adaptive- immune response. Upon antigen-specific activation, they can help B cells, influence the type of immune response via their cytokine secretion pro- file, or engage in cytolytic activity. Approximately three-quarters of pe- ripheral blood mononuclear cells are T lymphocytes. Among them, CD4⁺ cells often referred to as T helper cells predominate over CD8⁺ cells. Numerous studies have reported that enhanced apoptosis causes a remarkable loss of CD4⁺ and CD8⁺ T cells in protracted sepsis [10,38,39]. In the current study, both CD4⁺ and CD8⁺ T cells increased significantly undergoing SFI therapy (Fig. 2A and B), which indicated

APACHEIIscore on day 7	16.9 +- 8.8	13.2 +- 7.6	.034	that SFI might be able to induce CD4+ and CD8+ T-lymphocyte cell pro-
Marshall score on day 7	8.5 +- 3.3	6.8 +- 2.6	.010	liferation or prevent apoptosis during sepsis. However, significant differ-
Duration of vasopressor use (d)	3.7 +- 1.7	2.5 +-1.5	.008	ences in T-cell function were not noted between the 2 groups. In the

The SFI group was treated with SFI; the placebo group was treated with a placebo.

future, this should be investigated in more detail.

In addition, monocytes from patients with sepsis typically show a di- minished ability to release LPS-induced proinflammatory cytokines, such as TNF and IL-6. The proinflammatory cytokines are able to release anti-Inflammatory mediators, mainly IL-10, which are either unimpaired or enhanced [11,40-42] and lead to an increase in the release of immuno- suppressive mediators and a decrease in antigen presentation as a result of the reduced expression of mHLA-DR. Both are associated with a worse outcome of sepsis [1,43,44]. Low levels of mHLA-DR expression function as a surrogate marker of monocyte unresponsiveness [43,45] and as an independent predictor of nosocomial infection and mortality after sepsis [35,36]. Regarding the assessment of monocytic function, the results showed that treatment with SFI substantially restored mHLA-DR expression (Fig. 2C) and significantly increased LPS-induced monocytic proinflammatory cytokine (TNF-? and IL-6) release in patients with sepsis (Fig. 2D and E). However, previous studies showed that the release of proinflammatory mediators may induce hemodynamic insta- bility, end-organ dysfunction, and coagulation abnormalities [12,14], which led to strategies that aimed to treat sepsis by targeting proinflam- matory mediators. However, most of these approaches have failed. An im- portant reason for this might be that most patients with septic shock do not die of complications resulting from an overwhelming proinflammato- ry immune response but of secondary/opportunistic infections resulting from a severely suppressed immune response [12,37,46]. Furthermore, ginsenoside, a major active ingredient, has effects that include scavenging free radicals [21], inhibiting inflammatory mediators [20,22], suppressing cell apoptosis [18,23], and regulating the host immune response [24,25]. It is of great interest to further define the mechanism by which SFI improves the cellular immunity reaction.

The current study also showed the beneficial effects of SFI on the du-

ration of vasopressor use, severity of illness, and degree of organ dys- function, which indicated faster recovery from sepsis. This also translated into a shorter ICU stay in the SFI group. In addition, the 28- day all-cause mortality was lower in the patients who received SFI, al- though it did not reach statistical significance (Table 2). However, dur- ing the study, we did see 4 patients who had headaches after the first dose of SFI and 5 patients who had an increased heart rate on the first 2 days after the SFI treatment. Those symptoms returned to normal after 4 days. The serum levels of alanine aminotransferase, alanine ami- notransferase, total bilirubin, urea nitrogen, and creatinine were evalu- ated, and there were no obvious differences among the SFI-treated patients and those in the placebo group, which was concordant with previous studies [47,48].

Some limitations of this study demand further investigations. First, T-cell function and other indices reflecting monocyte immunity, such as phagocytosis and antigen presentation, were not analyzed in the cur- rent study. These indices need to be investigated in subsequent analy- ses. Another limitation is the lack of prior stratification of patients with septic shock based on their immune functional status. Moreover, the power of this study is limited by the single-center design and rela- tively small sample size, and although the 28-day all-cause mortality was observed, the study lacked a longer-term follow-up.

In conclusion, this preliminary study investigated the effect of SFI on the cellular immunity and clinical outcomes of patients with septic shock. We found that SFI had significant efficacy to restore monocyte immunosuppression in the patients with sepsis. Importantly, the SFI group showed better clinical outcomes than did the placebo group in the duration of vasopressor use, APACHE II score, Marshall score, and length of ICU stay. These findings suggest that SFI can enhance the cel- lular immunity of patients with septic shock and could be considered a promising adjunctive treatment for this patient population.

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