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Hydroxyethyl starch resuscitation downregulate pro-inflammatory cytokines in the early phase of severe acute pancreatitis: A retrospective study

In the present study, we investigated the effects of hydroxyethyl starch (HES) 130/0.4 on serum pro-inflammatory variables, immunologic variables, fluid balance (FB)-negative(−) rate and renal function in severe acute pancreatitis (SAP) patients. From October, 2007 to November, 2008, a total of 120 SAP patients were enrolled in this retrospective study. Fifty-nine patients in the HES group received 6% HES 130/0.4 combined with crystalloid solution for fluid resuscitation (HES group). In the control group, 61 patients received only crystalloid solution after admission. Interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α levels in serum were measured on days 1, 2, 4 and 8. The peripheral blood CD4+CD8+ T lymphocyte rates, serum BUN and Cr values were also measured on days 1, 4 and 8. Patients with FB(−) rates were recorded from day 1 to 8. Interaction term analysis (hospital stay and fluid resuscitation methods) based on mixed-effects regression model revealed significantly lower levels of IL-1 and TNF-α in the HES group compared with the control group. The difference in curve's risk ratio was not significant for IL-6, CD4+CD8+ T lymphocyte rate, BUN and Cr values (P>0.05). In the HES group, we detected a significantly higher rate of patients with FB(−) from day 4 to 8 (P<0.05). Thus, HES 130/0.4 resuscitation could decrease the IL-1 and IL-8 levels, shorten the duration of positive FB, and preserve the patient's immune status as well as renal function during the early phase of SAP.


Introduction
Severe acute pancreatitis (SAP) is an acute inflammatory process with variable involvement of local tissues and remote organs (1). The overexpression of inflammatory cytokines, such as interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α, can seriously damage the microcirculatory system's endothelium and subsequently increase capillary permeability (2,3). Additionally, the persistent inflammatory state aggravates the existing hypoxia condition and systemic inflammatory response syndrome (SIRS), which eventually result in an increase in the mortality rate associated with SAP (4,5).
In addition to the cytokine cascade, the activation of the adaptive immune system, including CD4 + CD8 + T lymphocytes is crucial in the development of SIRS and organ failure in SAP patients (6,7). Results obtained from prior studies reported a significant reduction in peripheral blood T-cell subgroups in the early phase of AP, which may correspond with patients' clinical outcome and disease severity (8,9). In a previous study, we reported that the reduction in CD4 + T lymphocytes in peripheral blood was linked to organ failure and may act as a potential predictor of severity in AP patients (10). Effective fluid resuscitation and immune regulation therapy is significant for the prognosis of SAP in the early phase.
Among patients with AP, early fluid resuscitation is often associated with a reduced incidence of SIRS and organ failure (11,12). Currently, two types of fluids are frequently used for active resuscitation: Crystalloid and colloid fluids with large molecules that maintain the fluid intravascularly. Hydroxyethyl starch (HES) is a widely used colloid fluid in volume expansion and is a priming fluid in the early stage Hydroxyethyl starch resuscitation downregulate pro-inflammatory cytokines in the early phase of severe acute pancreatitis: A retrospective study of AP (13,14). Previous findings have shown that HES resuscitation in the early stages of SAP can decrease the acute physiology and chronic health evaluation II (APACHE II) score, reduce the need for mechanical ventilation, reduce the risk of intra-abdominal hypertension, and shorten the duration of positive fluid balance (FB) (15,16). However, recent studies indicated that HES may not provide a clinical benefit and may even cause a higher mortality rate as well as higher acute kidney injury rate in critically ill patients (17)(18)(19)(20). This controversy aroused concerns regarding the safety of using HES resuscitation during the early phase of SAP. By contrast, other studies have shown that HES 130/0.4 can reduce the pro-inflammatory responses and regulate the CD4 + CD8 + T lymphocyte subgroup ratio in cardiac surgery (21,22). However, whether HES 130/0.4 is effective for the reduction of pro-inflammatory cytokines and safe for renal function was the focus of the present study.

Materials and methods
Data source. All relevant clinical data were extracted from the database of a multi-center, randomized controlled trial (RCT) study, which was designed to observe the effects of early goal-directed fluid therapy with HES 130/0.4 on intraabdominal hypertension (IAH), multiple organ dysfunction and FB in SAP patients. This RCT study was conducted from October, 2007 to November, 2008 and a total of 120 SAP patients from four sites were enrolled in the present study. There were no significant differences between the groups regarding demographic data (P>0.05) ( Table I).
Patients. Participants were adult patients (aged 18-65 years) admitted within 3 days of disease onset. The diagnostic and classification criteria included two of the following features: i) Abdominal pain consistent with AP; ii) amylase activity at least 3-fold greater than the upper limit of normal; and iii) characteristic findings of AP on contrast-enhanced computed tomography CT) and, less commonly, on magnetic resonance imaging or transabdominal ultrasonography. Patients with confirmed SAP were considered eligible. The patients had at least one of the following criteria: i) Failure in at least one organ as defined by the Atlanta classification; ii) an APACHE II score ≥8; and iii) a Balthazar's CT grade classification score ≥7 (23). Patients with the following conditions were excluded: i) History of allergy to HES 130/0.4; ii) serious cardiac or renal failure; iii) serum albumin value <25 g/l; iv) blood coagulation dysfunction with international normalized ratio >3; v) history of other colloid intravascular volume-replacement regimens within 3 months prior to their enrollment in this study; vi) pregnant or lactating female patients; and vii) MOF needing continuous renal replacement treatment after admission.
Treatments. All the patients received specialized medical therapy for AP according to the United Kingdom, Chinese Medical Association, and International Association of Pancreatology guidelines (24)(25)(26). After admission, patients were randomly divided into 2 groups. One group received crystalloid (the control group) and the other group received crystalloid plus HES 130/0.4 resuscitation (the HES group). Patients in the two groups were infused with lactated Ringer's solution at a basic rate of 1-2 ml/kg/h. The control group received only Ringer's lactate and saline solution for resuscitation. In the treatment group, 6% HES 130/0.4 (Voluven; Fresenius Kabi, Bad Homburg, Germany) was infused at a volume ratio of 1:3 compared with saline solution. The volume of HES was maintained at <50 ml/kg. The total rate and volume of intravenous fluid was controlled to maintain hemodynamic stability. A stable hemodynamic status was defined as a central venous pressure (CVP) of 8-15 mmHg (1 mmHg=0.133 kPa), a urine output >0.5 ml/kg/h, a mean blood pressure >65 mmHg, a heart ratio of 80-100/ min, a hematocrit >0.3 or a SpO 2 of CVP >0.7. Medication was planned to be administered for 7 days. After a negative FB emerged, the HES was disabled according to the patient's condition.
The study was approved by the Medical Ethics Committee of Union Hospital (Wuhan, China). The need for informed consent was waived by the Medical Ethics Committee because the study was a retrospective study using a database from which patients' identification information had been removed.

Measurement of parameters and detection methods.
Factors included in this study were: Age, gender, height, CT grade, APACHE II scores, white blood cell counts, serum ALT, AST, BUN, Cr, Ca 2+ , CRP and amylase. The intra-abdominal pressure (IAP) and CVP was measured at 10:00 a.m., 12:00 and 2:00 p.m. on the 1st day after admission according to the standard techniques established by the World Society of Abdominal Compartment Syndrome in 2006 (27). The mean IAP value was calculated and the CVP measurement protocols were defined as previously described (28).
Blood samples were obtained on days 1, 2, 4 and 8 after hospitalization and serum pro-inflammatory mediators including IL-1, IL-6, IL-8 and TNF-α levels were measured using enzyme-linked immunosorbent assays (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Additionally, patients' blood samples on days 1, 4 and 8 were used to detect the rate of CD4 + CD8 + T lymphocytes. The measurement protocols were described in a previous study (10).
Statistical analysis. Empower (R) (www.empowerstats.com; X&Y Solutions, Inc., Boston, MA, USA) and R (http://www.Rproject.org) software were used for the statistical analysis. Missing data were described as 'NA' as per Empower (R) requirements. Data were presented as mean ± SD or proportions. Comparisons between groups were performed using the Chi-square test for categorical variables and two-sample t-test for continuous variables. P<0.05 was considered to indicate a statistically significant difference.
We compared the inter-group differences with regard to the patients' general condition, CT grade, APACHE II score, IAP value after admission, peritoneal drainage rate, inflammatory cytokine value, lymphocyte subsets rates and other biochemical values. In order to obtain a clear description of changes, we made line charts using the inflammatory cytokine values (including IL-1, IL-6, IL-8 and TNF-α) on days 1, 2, 4, and 8 after admission. Line charts were also made using lymphocyte subset rates (including CD4 + CD8 + ) on days 1, 4, and 8 after admission.
We evaluated the effect of the treatment on inflammatory cytokine levels and lymphocyte subset rates with regression analyses in the two groups. Additionally, a model adjusted for potential confounders [gender, age and peritoneal drainage (yes or not)] was established simultaneously. The longitudinal changes in inflammatory cytokine values and lymphocyte subset rates were analyzed with linear mixed-effects regression models in the two groups. We also calculated the effect of HES 130/0.4 on the serum BUN and Cr values based on the abovementioned model.
Changes in the inflammatory cytokine levels and lymphocyte subset rates were dependent on the treatment options and curing time. To reveal whether the effect of HES 130/0.4 was critical for the longitudinal changes in the inflammatory cytokine values and lymphocyte subset rates, a mixed-effects model was set-up to estimate the interaction between a fixed effect variable and hospital stay. First, the variable data were transformed into multiple records based on the admission time. Potential confounders [gender, age and peritoneal drainage (yes or not)] were also adjusted in these models. Then, the treatment effect (crystalloid plus HES 130/0.4 resuscitation or only crystalloid resuscitation), time effect (hospital stay) and the interaction between the treatment and time effects in both groups were evaluated in multiple regression models. Statistical analysis (P) elicited whether the fixed effect variable of the HES group was significant for the inflammatory cytokine values and lymphocyte subset rates when compared with the control group. We also calculated the effect of HES 130/0.4 on the serum BUN and Cr values based on the above model.

Results
Scatter plots of CD4 + CD8 + T lymphocyte rates and inflammatory cytokine value. Fig. 1 shows the difference in the lymphocyte subset rates and inflammatory cytokine levels between the two groups. CD4 + CD8 + T-cell rates were comparable on days 1 and 8 after hospitalization, whereas the CD4 + CD8 + T rate was higher on day 4 after hospitalization ( Fig. 1A and B). Lower levels of IL-1, IL-6 and IL-8 were detected in the HES group compared with the control group on days 1, 2, 4 and 8 after hospitalization (Fig. 1C-E). Higher TNF-α level was detected in the HES group on day 1 (Fig. 1F).
We also observed lower level of TNF-α on days 2, 4 and 8 in the HES group compared with the control group (Fig. 1F). In model I, IL-1, IL-6, IL-8 and TNF-α levels were reduced significantly in the HES group, while IL-6 and IL-8 Table II. Regression analyses of the changes of CD4 + CD8 + T lymphocyte rates, inflammatory cytokine and renal function values based on different analysis models. Additionally, its impact on the changes in the BUN and Cr values was analyzed in this model. Potential confounders, including gender, age and peritoneal drainage (yes or no), were also adjusted in this model. As shown in Table III, there was a significant elevation in the CD4 + T lymphocyte rates [risk ratio (RR) difference = 0.725, P<0.001] and reduction in the IL-6, BUN and Cr levels  Table III. Regression analyses of the impact of hospital stay, treatments and the interaction between hospital stay and different fluid resuscitation methods for the changes of CD4 + CD8 + T lymphocyte rates, inflammatory cytokine and renal function value. Percentage of patients with negative FB at 8 days. We calculated the number of patients with negative FB(-) on a daily basis. Patients with peritoneal drainage were not included in this analysis. After excluding those patients with peritoneal drainage, 50 cases remained in the HES group and 51 cases in the control group. As shown in Table IV, there was a significantly higher number of patients with a negative FB from day 4 to 8 in the HES group (P<0.05).

Discussion
In the early phase of SAP, activation of inflammatory cells leads to the release of pro-inflammatory mediators which lie at the heart of the pathologic process and are involved in all aspects of the cascade leading to SIRS and multiple organ dysfunction syndrome (29). It has been established that the excessive production of inflammatory mediators is responsible for the escalation of localized pancreatic inflammation into a generalized systemic inflammatory response, irrespective of the initiating stimulus (30). Many studies have shown that plasma levels of pro-inflammatory cytokines are usually elevated early in the course of AP and are associated with severity of the disease (2,(31)(32)(33)(34). Thus, resolving the inflammatory status by downregulating the pro-inflammatory cytokines significantly improves the prognosis. HES is a type of colloid solution that is widely used for fluid resuscitation in intensive care units. HES has the advantages of minimizing resuscitation volumes and the potential to sustain the intravascular volume for longer periods (35). Results obtained from our previous studies suggested that fluid resuscitation with HES in the early stages of SAP could improve the prognosis (16,36). Additionally, a recent study has indicated that HES resuscitation may attenuate SIRS by downregulating pro-inflammatory cytokine (21). However, the exact mechanism for the HES effect on cytokines is not well understood. Working on rat sepsis models, Feng et al reported that HES 130/0.4 inhibited the activation of nuclear factor-κB and neutrophil adhesion and migration, thus inhibiting cytokine production (37). Schäper et al demonstrated that HES prevented the inflammatory reaction by relieving ischemia reperfusion injury in the intestine (38). In the present study, we have shown that HES combined with crystalloid fluid resuscitation decreased IL-1 and TNF-α levels in peripheral blood.
IL-1 is an important mediator of inflammatory changes during pancreatitis. During the early phase of AP, IL-1 initiates the inflammatory cascade and activates the endothelium, allowing the migration of neutrophils into the post-venule and resulting in neutrophil degranulation, adhesion molecule expression, and chemokine activity. Additionally, TNF-α derived from macrophages and monocytes interacts with a number of other cytokines such as IL-1, IL-6 and platelet activation factor, which participate in this process simultaneously (39). The present study demonstrated that IL-1 and TNF-α levels decreased significantly in the HES group (P<0.05) while the IL-8 level decreased only marginally in this group compared with the control group (P=0.054). These results suggested that HES combined with colloid fluid resuscitation decreased the pro-inflammatory cytokine concentration and improved the SIRS status.
In addition to the pro-inflammatory cytokine cascade, the activated adaptive immune system including CD4 + CD8 + T lymphocytes are central to the development of SIRS and organ failure in AP patients (40,41). Previous findings have shown that a significant reduction in the proportion of CD4 + T cells is correlated with the severity of AP (8,9,42). In a previous study, we showed that the reduction of peripheral blood CD4 + T lymphocytes was associated with persistent organ failure (10). Ozturk et al reported a higher CD4 + T lymphocyte level and CD4 + :CD8 + T-cell ratio, in coronary surgery patients, in the HES 130/0.4 group compared with the crystalloid group (21). Differences in the CD4 + CD8 + lymphocyte subset rates between the HES and control groups were not significant in this study. This phenomenon may be explained by the fact that the immune system in SAP patients is affected by multiple organs and colloid resuscitation alone is insufficient to influence patients' adaptive immune system. The mechanism by which HES may affect CD4 + CD8 + lymphocyte subsets is still unclear.
Early effective fluid resuscitation is recommended to shorten the duration of SIRS and reduce morbidity and mortality among AP patients (43). However, higher capillary permeability results in loss of fluid from the intravascular space and fluid sequestration into the third space, which facilitates the deficiency in blood volume. Excess fluids may be harmful for effective organ perfusion, in critically ill patients and can increase the mortality rate and cause various complications, including IAH and abdominal compartment syndrome, which are associated with a poor prognosis for SAP patients (44). Previous findings have shown that FB-positive(+) status was associated with the poor prognosis of critically ill patients (45,46). Barmparas et al reported that the early attainment of FB(-) status was associated with a nearly 70% reduction in the risk of mortality in critically ill surgical patients (47). Therefore, maintaining colloid osmotic pressure and achieving FB(-) status earlier are important factors for the prognosis of SAP patients. This study presented significantly higher rates of patients with FB(-) from day 4 to 8 in the HES group after excluding those patients with peritoneal drainage, which indicated HES 130/0.4 combined crystalloid resuscitation could significantly shorten FB(+) duration. This can be explained by the fact that HES 130/0.4 belongs to a family of polydispersed colloid solutions with polymerized amylopectin molecules which do not leak from capillary vessels. This characteristic makes HES to sustain its colloid osmotic pressure longer than crystalloid solutions alone (48). These results also suggested that HES combined with crystalloid fluid resuscitation could negatively affect the release of pro-inflammatory cytokines, which may be another cause for the effect of HES on FB.
Recently, safety concerns for the clinical use of HES 130/0.4 for acute volume resuscitation have attracted the attention of the researchers in this field. The most important concern was the effect of HES 130/0.4 on renal function (17,18,20). However, we did not observe any HES related effects on renal function for SAP patients in this study. However, no patients with renal failure were included in this study. Due to potential acute kidney injury, HES 130/0.4 should be used with extreme caution for SAP patients with renal failure.
There are certain limitations to our study. First, this was an observational study that revealed the effect of HES 130/0.4 on pro-inflammatory cytokines, FB status and renal function. Whether HES 130/0.4 may provide a better prognosis merits further investigation. Second, our study only included data obtained from the first 8 days after hospitalization. Third, previous studies reported that HES 130/0.4 changed the CD4 + T lymphocytes and the CD4 + :CD8 + T cell ratio compared with those patients who only received crystalloid resuscitation after coronary surgery (21). However, we did not observe this effect in this study. This may be due to the fact that HES 130/0.4 was only used in the 1st week, which did not produce any changes in patient's immune status. The exact mechanism for the effect of HES 130/0.4 on CD4 + :CD8 + T lymphocytes should be investigated in future.
In conclusion, we identified that HES treatment could decrease IL-1 and IL-8 levels, shorten the duration of positive FB, and preserve the patient's immune status and renal function during the early phase of SAP.