A Systematic Review of Rhubarb (a Traditional Chinese Medicine) Used for the Treatment of Experimental Sepsis

Sepsis is a global major health problem in great need for more effective therapy. For thousands of years, Rhubarb had been used for various diseases including severe infection. Pharmacological studies and trials reported that Rhubarb may be effective in treating sepsis, but the efficacy and the quality of evidence remain unclear since there is no systematic review on Rhubarb for sepsis. The present study is the first systematic review of Rhubarb used for the treatment of experimental sepsis in both English and Chinese literatures by identifying 27 studies from 7 databases. It showed that Rhubarb might be effective in reducing injuries in gastrointestinal tract, lung, and liver induced by sepsis, and its potential mechanisms might include reducing oxidative stress and inflammation, ameliorating microcirculatory disturbance, and maintaining immune balance. Yet the positive findings should be interpreted with caution due to poor methodological quality. In a word, Rhubarb might be a promising candidate that is worth further clinical and experimental trials for sepsis therapy.


Introduction
Sepsis is a clinical syndrome resulting from an inflammatory response to a severe infection. It is characterized by systemic manifestations of infection, including vasodilation, leukocyte accumulation, and increased microvascular permeability in tissues remote from the infection. Nowadays, it is a global major health problem with great incidence rates, high mortality rates, and huge cost consumption. Although the guidelines of "Surviving Sepsis Campaign" have led to advances in sepsis therapy and thus a reduction in sepsis mortality [1], the mortality rates are still high, and there is an absolute necessity for more effective therapy [2].
In the course of human history, infectious diseases have always been a major subject, and sepsis was a whole new concept extracted from that in the 1990s, but it was never a new thing to human beings as to the disease itself [2] and it must be even more common in the ancient times due to poor environmental sanitation and lack of modern medical technology. Since ancient times, great efforts have been made to struggle with sepsis in China. The oldest and greatest existing Chinese medical classic, Huangdi's Internal Classic (Huang Di Nei Jing), first recorded different sepsisrelated symptoms. Treatise on Febrile Diseases (Shan Han Lun), written in the Eastern-Han dynasty, is the oldest existing Chinese medical monograph concerning infectious disease. In the Jin and Yuan dynasties, seasonal febrile disease theory formed and developed, further improving the Chinese clinical science of febrile diseases.
Rhubarb (Da Huang), one of the most popular traditional Chinese medicines used to control various diseases for thousands of years, is made of roots and rhizomes of Rheum palmatum L., Rheum tanguticum Maxim. ex Balf., Rheum undulatum, or Rheum officinale Baill. [3,4]. It was first documented in Shen Nong's Herbal Classic (Shen Nong Ben Cao Jing), the oldest Chinese materia medica, and later in Huangdi's Internal Classic (Huang Di Nei Jing) and was commonly employed in Treatise on Febrile Diseases (Shan Han Lun) and seasonal febrile disease theory as a purgative and bactericidal agent to reduce fever, promote blood circulation, and cleanse the body. In the past decade, pharmacological research revealed its potential to be applied to infectious disease for its numerous pharmacological activities such as anti-inflammatory [5][6][7], antimicrobial [3,8], antifungal 2 Evidence-Based Complementary and Alternative Medicine [3,8,9], antivirus [10], and immunoenhancing [3]. Recent studies have further researched Rhubarb as a treatment for sepsis in humans and animal models. However, assessment of efficacy and mechanisms of Rhubarb in treating sepsis still lack systematic analysis. Herein, we report a systematic review of the use of Rhubarb in experimental sepsis in this paper. The objectives of this study were to (1) systematically review and assess the experimental evidence for Rhubarb administered before or after onset of sepsis in animal models; (2) determine the efficacy of Rhubarb in sepsis and explore the impact on the efficacy of defined characteristics; (3) analyze the possible antisepsis mechanisms of Rhubarb; (4) propose the development for the design of future experimental sepsis and ultimately for further clinical trials in human patients.

Search Strategy.
We identified studies of Rhubarb in animal models of sepsis from PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, EMBASE, Chinese National Knowledge Infrastructure (CNKI), VIP Database, Wanfang Data, and Chinese Biomedical Literature Database (CBM) by using the terms "Rheum" OR "Rhubarb" OR "emodin" OR "rhein" OR "Dahuang" AND "sepsis" OR "septicemia" OR "septic shock" OR "endotoxic shock" OR "toxic shock" OR "blood stream infection" in English or in Chinese (the search terms used in PubMed are listed in the appendix). All searches were limited to studies on animals without language restrictions from the inception of each aforementioned database up to December 2014. Reference lists of all included articles and relevant reviews were also handsearched. Two authors (Yan-na Weng and Jing-xia Zheng) identified studies from databases independently. Disagreements were solved after discussion with a third party (Yun Han). If the data were missing or conflicting, we tried to get further information by contacting the authors. The study would be excluded if no further information is available and the key data remain conflicting. Two authors (Yan Zhang and Jiong-dong Du) extracted data independently. Disagreements were solved after discussion with a third party (Yun Han).

Study
Selection. 2394 papers were retrieved from seven databases. 70 articles remained after screening by titles and abstracts, of which 31 records were removed for being duplicates. Another 3 articles were excluded for no full text was available with effort. By full-text reviewing, 12 studies were excluded according to the exclusion criteria. Three more eligible studies were included after handsearching of reference lists of the included articles and other relevant reviews. Finally, a total of 27 studies were included in qualitative synthesis . The study selection process is summarized in a flow diagram (Figure 1).  1992 and 2014. 14 of the 27 trials were within the last 5 years. All were conducted in China.

Sample
Sizes. The design number of animal in TG (treatment group) for each outcome index at each time phase varied from 5 to 15, while 2 articles [17,31] did not report the design number of animals in each subgroup. 10 articles set Table 1: Model details of included trials.

Rhubarb Intervention.
Six studies [27,34,37,[39][40][41] used emodin, one of the active ingredients in Rhubarb, as the intervention for TG. The dosage varied from 20 mg/kg to 60 mg/kg per time and from 20 mg/kg to 300 mg/kg in total during course of treatment. Three studies [27,40,41] were intragastric (IG) administration, 2 studies [37,39] were IP, and 1 study [34] was enema. Only one study [33] used rhein, another kind of active ingredients in Rhubarb, as treatment for TG. The rest 20 studies all reported using rhubarb in TG; however, none of these studies tested the actual active ingredients of the Rhubarb therapy they used. Three articles [15,28,32] reported using raw Rhubarb, while the remaining 17 articles did not mention the maturity of Rhubarb. Powder [20,22,24,28,32,35] form of Rhubarb was used in 6 studies, decoction [15,25,31] and extract (no available details) [17,21,23] of Rhubarb were used in 3 studies, respectively, granule [30,36] [15,17] did not describe statistical method. One article [34] reported all the outcomes graphically, while 12 articles [16, 17, 21-26, 29, 33, 37, 41] reported partially in graphical way and partially in numerical way, and the rest 14 articles reported all the data numerically. Only one study [17] completed followup of animals' survival rates up to 40 days, and none of the 27 studies utilized intention-to-treat analysis when dealing with data of animals that died long before samples could be taken according to schedule. The checklist for study quality and risk of bias are shown in Table 2.

Effectiveness.
Meta-analysis could not be carried out due to the high heterogeneity and low methodological quality of the studies.

Effects of Rhubarb on Gastrointestinal Biological Barrier in Experimental Sepsis.
Three studies [29,31,32] reported significant effects of Rhubarb on restoring balance of gastrointestinal microflora during sepsis, manifesting in less loss of normal gastrointestinal flora (such as Bifidobacterium [31] and normal colibacillus [29]) and less growth of opportunistic enteropathogenic bacteria (such as Escherichia coli) [31,32].

Effects of Rhubarb on Gastrointestinal Mechanical Barrier in Experimental
Sepsis. Rhubarb therapies were reported significantly reducing injuries of gastrointestinal mucosa [16,18,19,26,27,35], decreasing intestinal microvascular permeability [18,19,26,27], improving microcirculation [27,34,36], and ameliorating metabolism [36] of intestine during sepsis. Resistance to apoptosis of intestinal epithelial cells in sepsis animals treated with Rhubarb was also described [26]. Lower incidence of bacterial translocation to remote organs [16,29] during severe sepsis might be a result of the effects of Rhubarb on gastrointestinal biological and mechanical barrier.

Effects of Rhubarb on Liver Injury in Experimental
Sepsis. Chen [24] reported that Rhubarb therapy could protect liver from injury during sepsis. The effects of reducing the loss of liver mitochondria cytochrome C ATP and liver mitochondria cytochrome oxidase, and inhibiting inflammation by suppressing mRNA expression of inflammatory cytokines (TNF-and IL-1 ) and their receptors, might be the mechanism of the protective effect.
The detailed outcome indexes of included studies are summarized in Table 3.

Discussion
The present study is the first systematic review of Rhubarb for animal model of sepsis in both English and Chinese literatures. The present study showed that Rhubarb might be effective in reducing injuries induced by sepsis in gastrointestinal tract, lung, and liver, and its potential mechanisms for 6 Evidence-Based Complementary and Alternative Medicine  [15] + antisepsis might include reducing oxidative stress and inflammation, ameliorating microcirculatory disturbance of adhesion molecule expression, leukocyte adhesion and cytokine release, and maintaining immune balance during sepsis. However, there was significant heterogeneity among the included studies, which might affect the effectiveness of Rhubarb therapy. From analysis of the heterogeneity, we can conclude several implications for further research.
Firstly, heterogeneity of Rhubarb intervention treatment was one of the most important factors.
The animal model for sepsis in the included studies mainly lies in LPS injection model (including LPS+ model like scald plus LPS injection) and CLP model. LPS model is notable for the advantages of simple, reproducible, highly controlled, and standardized administration, while, at the same time, the disadvantages of it cannot be easily overlooked: early, high, and transient increases of inflammatory mediators through a TLR4-dependent pathway, different hemodynamic response from human sepsis, not being able to reflect all complex physiological responses in human sepsis, and variability in dose and administration route [53][54][55][56][57]. The literature indicated that LPS administrated intraperitoneally induced systematic responses mainly via the lymphogenous route rather than the hematogenous route. Portal LPS can be effectively eliminated by the liver and does not reach the systemic circulation at all unless the LPS concentration is extremely high. Besides, intraperitoneal administration of LPS is transported mainly in lipoprotein-bound form, which is less active in inducing systematic responses [58]. It had been reported that the dose of intraperitoneally administered LPS was approximately 100 times of the intravenous dose in order to produce a similar peak rise in temperature in New Zealand White rabbits [59]. However, the effects of different LPS administration doses or routes on course or outcomes of sepsis still deserve further investigation.
Comparing to LPS model, CLP model is characterized by its similar hemodynamic, immune, and metabolic phases to human sepsis by recreating human polymicrobial sepsis progression. The CLP model has even been titled with "the golden model" for sepsis [55,57]. Yet the complexity of human sepsis still cannot be completely reproduced by CLP model and the severity varies by differences in model procedures [55][56][57], including needle size for puncture, number of cecal punctures, type of suture, operation incisions, and volume of cecal contents extruded [57,60].
Pentobarbital, urethane, chloral hydrate, and ketamine are commonly used anesthetic drugs in animal experiment research. Studies demonstrated that these anesthetics had significantly different effects on systemic hemodynamics [61,62], organ functions, and immune response [63,64] in experimental animals. It was reported that chloral hydrate (300 and 400 mg/kg) severely depressed the cardiovascular and respiratory systems in rats, while pentobarbital (40 mg/kg) produced a moderate to severe depression and urethane (1.2 and 1.5 g/kg) was at a moderate level [65]. Urethane induced apoptosis in kidney, while pentobarbital had little effect on renal cell apoptosis [66]. Although pentobarbital augmented LPS-induced hypotension, it also attenuated LPSinduced acute lung injury (ALI) and organ dysfunctions [67]. Ketamine inhibited hypotension, suppressed cytokine responses, and reduced organ dysfunction in sepsis animals in vivo [68,69], but its depression on cardiac function of isolated rat hearts was significantly greater than pentobarbital and chloral hydrate [70]. Reevaluation should be taken into consideration when interpreting the results of experiments anesthetized with these anesthetic agents. Besides, it is worth noting that, in experimental sepsis, there have been no comprehensive comparisons among all these anesthetics. Which anesthetic is the most suitable agent for sepsis experiments remains unknown.
Meanwhile, as the elderly population is the major population of sepsis and age is an independent predictor of mortality, aged animals should be under consideration for experimental sepsis [53,56]. Likewise, comorbidities that alter cardiovascular and immune function should be involved [56]. Also, gender selection is another crucial element since estrogen was found to be protective in immune function during experimental sepsis [56]. Not to mention the environmental controls and medication (including supportive therapies) that might affect outcomes of sepsis animals should also be taken into consideration to mimic that of human sepsis [56].
Besides, neotype sepsis models has been invented to reproduce various sepsis physiological progressions [54,71]. So further studies for experimental sepsis should take the characteristics of different sepsis animal model into consideration and choose the most appropriate model.
Thirdly, quality of experimental design and reporting should be optimized.
A thorough protocol published before performance of experiments could reduce the risk of selective reporting. Journals can offer experiment reporting guidelines and carry out more restrictive policies for articles publication to help improve reporting quality. Statement of animal welfare compliance and sample size calculation are not criteria for risk of bias in animal trials, but they are important characteristics for evaluating the quality of evidence [11]. It is reported that risk of observer bias exists when animal model experiments lack blinding of outcome assessors [72]; however, experimental studies can almost always be blinded from allocation to model induction, intervention administration, and outcome assessment. Randomization process should be well designed and in detail reported to provide a more reliable basis for translational medicine from "bench to bedside." Numerical data, both in animal numbers (no matter in Method section or Result section) and outcome measure results, should be advocated as it is more valuable for further assessment or reanalysis of the results. Furthermore, reasonable design of animal group, completeness of follow-up, and objective process of incomplete data are crucial elements for a sound experiment design.
Moreover, there are several limitations in this systematic review. First, the literatures of languages other than Chinese and English were not included in this systematic review, to some extent that might result in selective bias. Second, most of the studies were published articles (24 out of 27 studies); all of the data were collected from the published papers without more information accessed from the authors after trying to contact with them. As a result, the efficacy of Rhubarb might be overestimated due to publication bias. Third, general methodological quality of included studies was poor, indicating that caution was needed when interpreting the results.

Conclusions
There is no similar systematic review on Rhubarb for sepsis. 27 studies were identified from 7 databases in this systematic review to evaluate the efficacy of Rhubarb therapy for sepsis. Rhubarb has been apparently reported to be effective in reducing injuries induced by sepsis. Yet the positive findings should be interpreted with caution due to poor methodological quality. Rhubarb might be a promising candidate that is worth further clinical and experimental trials for sepsis.