**Anil Kumar Chauhan, Jieun Kim, Yeongjoon Lee, Pavithra K. Balasubramanian and Yangmee Kim \***

Department of Bioscience and Biotechnology, Research Institute for Bioactive-Metabolome Network, Konkuk University, Seoul 05029, Korea; chauhananil48@konkuk.ac.kr (A.K.C.); za3524@konkuk.ac.kr (J.K.); lyj7956@konkuk.ac.kr (Y.L.); Pavithra@konkuk.ac.kr (P.K.B.)

**\*** Correspondence: ymkim@konkuk.ac.kr; Tel.: +822-450-3421; Fax: +822-447-5987

Academic Editors: H.P. Vasantha Rupasinghe and Vincenzo De Feo Received: 9 September 2019; Accepted: 31 October 2019; Published: 4 November 2019

**Abstract:** Isorhamnetin is a flavonoid that is abundant in the fruit of *Hippophae rhamnoides* L. It is widely studied for its ability to modulate inflammatory responses. In this study, we evaluated the potential of isorhamnetin to prevent gram-negative sepsis. We investigated its efficacy using an *Escherichia coli*-induced sepsis model. Our study reveals that isorhamnetin treatment significantly enhances survival and reduces proinflammatory cytokine levels in the serum and lung tissue of *E. coli*-infected mice. Further, isorhamnetin treatment also significantly reduces the levels of aspartate aminotransferase, alanine amino transferase and blood urea nitrogen, suggesting that it can improve liver and kidney function in infected mice. Docking studies reveal that isorhamnetin binds deep in the hydrophobic binding pocket of MD-2 via extensive hydrophobic interactions and hydrogen bonding with Tyr102, preventing TLR4/MD-2 dimerization. Notably, binding and secreted alkaline phosphatase reporter gene assays show that isorhamnetin can interact directly with the TLR4/MD-2 complex, thus inhibiting the TLR4 cascade, which eventually causes systemic inflammation, resulting in death due to cytokine storms. We therefore presume that isorhamnetin could be a suitable therapeutic candidate to treat bacterial sepsis.

**Keywords:** isorhamnetin; flavonoid; bacterial sepsis; toll-like receptor 4; inflammation

#### **1. Introduction**

Inflammation is an innate immune response against microbial infection. However, if it persists for a long time, it can cause several fatal diseases, including sepsis. Sepsis is a clinical condition defined as a systemic inflammation in the body in response to microbial infection, which eventually leads to multiple organ failure. Sepsis is reported to be the leading cause of mortality across the globe, and thus the World Health Organization (WHO) devoted the year 2017 to prioritize this disease and its treatment strategies [1–3]. The organs and systems that are commonly affected by sepsis include the lung, abdomen, blood and kidneys, with associated incidences of 64%, 20%, 15% and 14%, respectively [3]. Pathophysiological studies also reveal that the activation of toll-like receptors (TLRs) by microbes or microbial peptides represents the principle mechanism underlying the development of sepsis [3,4].

TLRs are transmembrane receptors present in major immune cells, and are composed of an extracellular leucine-rich repeat domain, a transmembrane region and an intracellular toll-interleukin-1 receptor (TIR) domain [5,6].

In total, 10 and 12 TLRs are identifiable in human beings and mice, respectively, and each of them recognizes specific pathogen-associated molecular patterns [7]. However, among these TLRs, TLR4 is responsible for the recognition of bacterial lipopolysaccharide (LPS) and the initiation of an immune response against LPS and/or gram-negative bacteria. Notably, TLR4 requires MD-2 to recognize LPS, and a TLR4–MD-2–LPS complex is essential to activate the TLR4 pathway in macrophages. Once TLR4 recognizes LPS, it undergoes an oligomerization process to recruit downstream adaptor molecules via interactions with the TIR domain. Five adaptor proteins that contain the TIR domain are identifiable, including MyD88 (myeloid differentiation primary response gene 88), TIRAP (TIR domain-containing adaptor protein, also known as Mal, MyD88-adapter-like), TRIF (TIR domain-containing adaptor inducing IFN-β), TRAM (TRIF-related adaptor molecule), and SARM (sterile α and HEAT-Armadillo motifs-containing protein) [8]. These adaptor proteins play crucial roles in the TLR4 pathway, which eventually results in the translocation of NF-κB from the cytoplasm to the nucleus to trigger the production of pro-inflammatory cytokines [7].

Flavonoids are naturally-occurring polyphenols that are found in a variety of edible plants. Chemically, they exist in glycosylated (conjugated with sugar) or aglycone forms (free form) [9]. The role of flavonoids in plants is crucial, as they confer infection resistance to the plant [10], in addition to protecting them from harmful ultraviolet rays [10,11]. Moreover, much research was carried out to explore the role of flavonoids in animal systems, and among the various activities shown by all types of flavonoids, their antioxidant potential is the most notable [12]. They are also reported to have other health-promoting effects, such as anti-inflammatory activity. For example, the cytoprotective efficacy of plant flavanol quercetins is widely explored [13,14]. Isorhamnetin (Figure 1), another plant flavanol, is a 3- -O-methylated metabolite of quercetin, and is found predominantly in the fruit of *Hippophae rhamnoides* L.; it exerts various biological effects, including anti-inflammatory [15], anticancer [16] and antioxidant activities [17]. Further, we previously reported that this flavonoid has potent anti-tuberculosis activity against *Mycobacterium tuberculosis* H37Rv and multi-drug- and extensively drug-resistant clinical isolates [18]. Importantly, the absorption and metabolic stability of methylated flavonoids are believed to be higher than those of unmethylated flavanols [16]. Thus, this property makes isorhamnetin more potent than quercetin.

**Figure 1.** Chemical structure of isorhamnetin also known as 3- -methoxyquercetin (molecular weight: 316.26). The image was drawn using Chemdraw software.

In the present study, we explored the potential of isorhamnetin to protect against *Escherichia coli*-induced sepsis by establishing a murine model. Furthermore, based upon binding affinity and a molecular docking examination, we show for the first time, to our knowledge, that isorhamnetin can bind TLR4/MD-2 directly, thus preventing the activation of the TLR4 cascade, which is responsible for sepsis progression. The outcomes of our study might provide further insights into the natural flavonoid isorhamnetin and its role in sepsis prevention and treatment.

#### **2. Results**

#### *2.1. Isorhamnetin Treatment Protects Mice from E. coli-Induced Sepsis*

It was previously reported that isorhamnetin can reduce inflammation by downregulating nitric oxide (NO) and cytokine production [19]. Therefore, we sought to examine the ability of isorhamnetin to protect mice from *E. coli*-induced sepsis. To establish a bacterial sepsis model, we used a virulent *E. coli* K1 strain to infect isorhamnetin-pretreated mice for 96 h for survival assays and 18 h to examine inflammation. As depicted in Figure 2A, mice in the isorhamnetin-treated group survived until the end of the experiment, but the *E. coli*-treated mice died within 24 h of infection. Further, we assessed the bacterial burden in the visceral organs of the mice (lung, liver, and kidneys) and found that isorhamnetin treatment significantly reduced the bacterial population in all organs compared to that in the *E. coli* only group (Figure 2B). Although isorhamnetin has no antibacterial activity in vitro, it is reported to enhance macrophage functions, such as phagocytosis, by increasing superoxide generation after the engulfment of bacteria [20]. Thus, we speculate that the reduction in bacterial loads in the organs is a result of the immunomodulatory potential of isorhamnetin.

**Figure 2.** In vivo examination of the effects of isorhamnetin on *Escherichia coli*-induced sepsis. (**A**) Potential of this compound to promote the survival of mice in response to *E. coli*-induced sepsis. (**B**) Evaluation of the bacterial load in the visceral organs of mice. Data are presented as the means ± standard error of the mean (SEM). \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001 compared to the *E. coli* group.
