Anti-influenza A Virus Effects and Mechanisms of Emodin and Its Analogs via Regulating PPARα/γ-AMPK-SIRT1 Pathway and Fatty Acid Metabolism

The peroxisome proliferator-activated receptor (PPAR) α/γ-adenosine 5′-monophosphate- (AMP-) activated protein kinase- (AMPK-) sirtuin-1 (SIRT1) pathway and fatty acid metabolism are reported to be involved in influenza A virus (IAV) replication and IAV-pneumonia. Through a cell-based peroxisome proliferator responsive element- (PPRE-) driven luciferase bioassay, we have investigated 145 examples of traditional Chinese medicines (TCMs). Several TCMs, such as Polygonum cuspidatum, Rheum officinale Baillon, and Aloe vera var. Chinensis (Haw.) Berg., were found to possess high activity. We have further detected the anti-IAV activities of emodin (EMO) and its analogs, a group of common important compounds of these TCMs. The results showed that emodin and its several analogs possess excellent anti-IAV activities. The pharmacological tests showed that emodin significantly activated PPARα/γ and AMPK, decreased fatty acid biosynthesis, and increased intracellular ATP levels. Pharmaceutical inhibitors, siRNAs for PPARα/γ and AMPKα1, and exogenous palmitate impaired the inhibition of emodin. The in vivo test also showed that emodin significantly protected mice from IAV infection and pneumonia. Pharmacological inhibitors for PPARα/γ and AMPK signal and exogenous palmitate could partially counteract the effects of emodin in vivo. In conclusion, emodin and its analogs are a group of promising anti-IAV drug precursors, and the pharmacological mechanism of emodin is linked to its ability to regulate the PPARα/γ-AMPK pathway and fatty acid metabolism.


Introduction
Highly pathogenic influenza A virus (IAV) infection or seasonal IAV infection of patients with basal metabolic diseases usually leads to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). And unfortunately, now, there is no specific medicine available for the treatment of ALI/ARDS. In addition, it is well known that it is difficult to control IAV infection through vaccination due to the rapid antigenic variation through "antigenic drift" and "antigenic shift." And furthermore, classical anti-IAV drugs, such as M2 channel and neuraminidase inhibitors, are also limited in use by their side effects or the resistant viral strains [1]. In consequence, the development of novel anti-IAV drugs is still an urgent need.
To research the synergistic effects of traditional Chinese medicines (TCMs) and their active compounds, we have engaged in the classification of TCMs according to the different IAV pathogenic mechanisms for many years. In recent years, the peroxisome proliferator-activated receptor (PPAR) α/γ signaling pathway has been shown to be an antiviral innate immune signal that can protect against lethal IAV attacks in mice [2,3]. It has been reported that PPARα agonist gemfibrozil can increase the survival of IAV (H2N2)-infected mice from 26% to 52% [2]. PPARγ agonists rosiglitazone, pioglitazone, and 15d-PGJ2 can reduce viral titer and protect mice from lethal IAV infection [3].
In addition, it has been reported that energy metabolic disorder, also named "mitochondrial energy crisis," is a major risk factor for severe IAV infection [9]. During the late phase of IAV infection, new treatment options have been proposed to target the energy crisis by restoring glucose and long-chain fatty acid oxidation, rather than antiviral treatments with neuraminidase inhibitors [9]. It has been reported that fatty acid metabolism is very important for the replication of many viruses. IAV infection can impair fatty acid oxidation [10] and upregulate fatty acid biosynthesis by increasing the levels of acetyl-CoA carboxylase (ACC) and fatty acid synthetase (FAS), which are two important enzymes for de novo fatty acid biosynthesis; ACC inhibitor 5-tetradecyloxy-2-furoic acid (TOFA) and FAS inhibitor C75 can inhibit IAV replication by >1,000-fold and >10fold, respectively [11,12].
PPARα/γ pathways play important roles in energy metabolism. PPARα agonists can increase the expression of fatty acid oxidation enzymes, improve mitochondrial membrane potential (ΔΨm), restore the ATP level, and reduce virus production [13]. In the recent study, based on a PPAR response element luciferase reporter (pPPRE-luc), we have screened 145 examples of TCMs and found that several TCMs, such as Polygonum cuspidatum, Rheum officinale Baillon, and Aloe vera var. Chinensis (Haw.) Berg, had high activity. In the following research, we have investigated the anti-IAV effects and mechanisms of emodin (1,3,8-trihydroxy-6-methylanthraquinone) and emodin analogs, all of which are important compounds of these TCMs [14].  (2000). Each specimen was deposited in our lab. The extracts of TCMs were deposited and protected from light in a -20°C refrigerator. Emodin and emodin analogs (Supplementary Figure 1) were purchased from MedChemExpress Co., Ltd (New Jersey, USA). Compound C, MK886, and GW9662 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). All other chemicals were analytical reagent grade.

In Vitro Cell Infection and Plaque Assay.
In the in vitro cell infection assay, the viruses were pretreated with virus growth medium (VGM) containing the test drug for 2 h. VGM is a MEM medium, which contains 1 μg/mL TPCKtrypsin and 3.2% bovine serum albumin. After pretreatment, the viruses were washed with PBS three times and concentrated by ultrafiltration. Before the experiment, MDCK or A549 cells were seeded into six-well plates for 24 h. Then, the pretreated viruses were added and adsorbed for 1 h (multiplicity of infection ðMOIÞ = 0:001) and washed with PBS three times, and the cells were cultivated in a series of mediums containing test drugs for 48 h. After frostthawing one time, the supernatants were collected and the titers were determined using a plaque assay, or the cells were used in qRCR or western assays.
In the plaque assay, MDCK cells were seeded into sixwell plates for 24 h. After washing with PBS three times, the cells were incubated with 0.2 mL of the collected supernatants (virus suspension) at 36°C for 60 min with frequent shaking. After discarding the supernatant and washing with PBS three times, a 0.6% agarose (1 mL) containing 1x VGM overlaid the plates. The plates were incubated at 36°C in a humidified atmosphere of 5% CO 2 in air. After 36 to 72 h, the plaques were stained with 1% crystal violet solution and counted.

Primary Screening of TCMs and Antiviral Assays.
In the primary screening of TCMs, A549 cells (4 × 10 4 ) were seeded in 96-well microplates for 24 h, then cotransfected with pPPRE-luc plasmid (Beijing Biolab Technology Co., Ltd., Beijing, China) and pRL-TK plasmid (internal control) using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, Carlsbad, USA). After 8 h, the cells were infected and treated with different drugs (including the positive drug (gemfibrozil) and the extracts of TCMs). After 24 h, the luciferase activity was determined following the instruments of the Luciferase Reporter Assay Kit (BD Biosciences Clontech, CA, USA). The Z ′ -factor, a statistical parameter to quantify the suitability of HTS, was calculated as previously reported [16]. In addition, to determine the antiviral with free access to food and water. After 1 week of adaptive feeding, the median lethal dose was first determined; then, the mice were randomly divided into 8 groups (n = 16 in each group). Before the experiment, the mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg). In the uninfected control (normal group), mice were not infected with IAV (PR8) virus but intranasally shammed with VGM medium and treated with PBS+DMSO (0.5%) by oral gavage. In the negative control (IAV group), positive control (oseltamivir, Ose group), and emodin-treated groups (EMO group), mice were intranasally infected with 50 μL IAV (PR8) virus solution (2:46 × 10 6 PFU) and orally treated with PBS+DMSO (0.5%), oseltamivir (10 mg/kg/day), and emodin (75 mg/kg/day) from -1 to 5 post infection (p.i.), respectively.
Ten mice from each group were observed for morbidity daily and weighed for 14 days. The other 6 mice of each group were put to death by dislocating their cervical vertebras on day 6 p.i. The lung index was assessed by determining the percent of lung wet weight (g) to body weight (g). Right lung tissues were frozen for western blot, qRT-PCR, ELISA, and viral titer assays, and left lung tissues were fixed in 10% neutral buffered formalin (NBF) for pathological analyses. The severity of histological changes was scored according to a semiquantitative scoring method [19].
2.11. Statistical Analysis. The statistical significance was assessed using SPSS16.0 software. Data were analyzed by one-way analysis of variance (ANOVA). The survival time was analyzed by Kaplan-Meier analysis with log-rank and Breslow tests. Results are expressed as the mean ± standard deviations ðSDÞ. P ≤ 0:05 was considered significant.

Results of Primary Screening and the Anti-IAV Effects of
Emodin and Its Analogs In Vitro. In this study, through a cell-based PPRE-driven luciferase bioassay, 145 examples of TCMs were investigated. As shown in Supplement Table 2, several TCMs showed high activity, such as Polygonum cuspidatum, Rheum officinale Baillon, and Aloe vera var. Chinensis (Haw.) Berg. After checking the pharmacology database of traditional Chinese Medicine (https://tcmspw.com/tcmsp.php) and the traditional Chinese medicine integrated database (https://tcm.scbdd .com/home/search_index/) as well as historical literary data [20], we found that emodin was an important compound 3 BioMed Research International of these TCMs. Followingly, we further discover the anti-IAV effects of emodin and its analogs, which are a group of anthraquinone compounds, existing in many plants and possessing antibacterial, antiviral, anti-inflammatory, and anticancer effects [21]. Before antiviral tests, we determined the cytotoxicity of emodin and its analogs. The results showed that emodin and its analogs had no significant cytotoxicity on A549 and MDCK cells below the concentration of 25 μg/mL (Supplement Figures 2 and 3). To detect their antiviral activity, we performed a plaque inhibition assay ( Figure 1) and a qRT-PCR assay (Supplement Figure 4). The results showed that emodin and its analogs could significantly suppress the replication of IAV (PR8) at concentrations from 12.5 to 25 μg/mL. In addition, several emodin analogs, such as emodin-1-O-β-D-glucopyranoside, chrysophanol-8-O-glucoside, aloe- isorhapontigenin, rhapontin, desoxyrhaponticin, and rhapontigenin 3 ′ -Oglucoside, still significantly reduced the replication of IAV even at the concentration of 3.125 μg/mL, which was better than emodin did.

PPARα/γ and AMPK Pathways Play Important Roles in
the Anti-IAV Effect of Emodin. Further, we chose emodin as our target compound to investigate the mechanism of action of these compounds. In this study, we first investigated the  Figure 2: Emodin activated the PPARα/γ-AMPK pathway in A549 cells. In the normal and emodin-(EMO-) treated groups, A549 cells were treated with DMSO (0.5% v/v) and emodin (25 μg/mL), respectively, but not infected with IAV. In the IAV-infected and IAV+Riband IAV+EMO-treated groups, A549 cells were infected with IAV (PR8, MOI = 0:001) and simultaneously treated with DMSO (0.5% v/v), ribavirin (Rib, 25 μg/mL), and emodin (EMO, 25 μg/mL), respectively. After 48 h, the mRNA levels of PPARα, PPARγ, and AMPK were quantified by a qRT-PCR assay (a). The protein levels of PPARα, PPARγ, AMPK, p-AMPK, and p-ACC were quantified by a western blotting assay by using ImageJ software. The results were expressed as the ratio of the target gene to β-actin (b, c). All data shown were the mean ± SD of three independent experiments. * P < 0:05 vs. the mock-treated group; # P < 0:05 vs. the only IAV-infected group. 5 BioMed Research International influences of emodin on the PPARα-AMPK signaling pathway. As shown in Figure 2, IAV infection could significantly increase the mRNA and protein expressions of PPARα/γ and AMPK and elevate the phosphorylation of AMPK and ACC in A549 cells. ACC is a major downstream substrate of AMPK, and the phosphorylation level of ACC can represent the serine/threonine protein kinase activity of AMPK. Emodin could further significantly increase the mRNA expression of PPARα/γ and AMPK and the phosphorylation of AMPK and ACC after IAV infection.    BioMed Research International To examine the significance of the PPARα/γ-AMPK pathway in emodin-mediated inhibition on IAV replication and IAV-induced injury of cell viability, specific inhibitors of PPARα (MK886), PPARγ (GW9662), and AMPK (Compound C, CC) were used. Through a qRT-PCR assay, we found that emodin-mediated inhibition on IAV replication was significantly counteracted by the treatments of MK886, GW9662, and CC (Figure 3(a)). Through a MTT method, we found that these inhibitors also significantly antagonized the inhibition of emodin on IAV-induced injury of cell viability (Figure 3(b)). Additionally, transfection of siRNAs for PPARα, PPARγ, and AMPKα1 also significantly counteracted the inhibition of emodin on IAV replication (Figure 3(c)) and on IAV-induced injury of cell viability (Figure 3(d)). These results indicated that the PPARα/γ-AMPK pathway was involved in the inhibition of emodin on IAV replication and IAV-induced cell injury.

Emodin Increased Fatty Acid Oxidation and Decreased
Fatty Acid Biosynthesis after IAV Infection. AMPK is a key cellular energy sensor, involved in lipid homeostasis and ATP balance regulation, and most of PPAR agonists exert their physiological effects through activating AMPK. We further determined the influence of emodin on the expressions of genes involved in fatty acid oxidation and biosynthesis. As compared with the IAV-infected control, emodin could further increase the expression of genes involved in fatty acid oxidation (such as carnitine palmitoyl-CoA transferase (CPT) IA, CPTII, acyl-CoA oxidase (ACOX) 1, and MLYCD) and decreased the expression of genes involved in fatty acid biosynthesis (such as SREBP-1c, ACC, and FAS) ( Figure 5).
Then, we determined the FAS activity, free fatty acid, and intracellular ATP levels using biochemical methods. The results showed that IAV infection significantly increased the activity of FAS and the level of free fatty acids and decreased the level of intracellular ATP, but emodin significantly reversed these biochemical changes ( Figure 6).
Further, we have also determined the significance of fatty acid oxidation and biosynthesis in emodin-mediated anti-IAV effect. We first determined the influence of β-oxidation inhibitors (CPT1 inhibitor etomoxir (ETO) and 3-ketoacyl coenzyme A thiolase inhibitor trimetazidine (TMZ)) on the inhibitory effect of emodin. As shown in Figure 7, unexpectedly, ETO and TMZ did not reverse the inhibition of emodin on IAV replication but further reduced the level of IAV vRNA. We further determined the effects of ETO and   Figure 4: Interference of PPARα/γ-AMPK-SIRT1 pathway impaired the inhibition of emodin on IAV-induced production of inflammatory cytokines. A549 cells were infected and treated as mentioned in Figure 2. The levels of cytokines were determined by the ELISA assay. All data shown were the mean ± SD of three independent experiments each in triplicate. * P < 0:01 vs. the only IAV-infected group; # P < 0:05 vs. the IAV+emodin group; Δ P < 0:05 vs. the IAV+emodin+siRNA control group.  Figure 5: Emodin regulated the gene expression of fatty acid oxidation and fatty acid biosynthesis in A549 cells. The treatments were the same as those in Figure 2. After 48 h, the mRNA and protein expressions were quantified by a qRT-PCR assay (a, b) and by a western blotting assay (c-e). All data shown were the mean ± SD of three independent experiments. * P < 0:05 vs. the normal group; # P < 0:05 vs. the only IAV-infected group. 8 BioMed Research International TMZ on IAV replication without emodin treatment and found that ETO and TMZ themselves could significantly inhibit IAV replication. Additionally, we further determined the influence of palmitate on IAV infection. Palmitate is the end-product of the de novo fatty acid biosynthesis; the addition of exogenous palmitate can be recognized as the activation of fatty acid biosynthesis. Our results showed that palmitate could impair the inhibitory action of emodin on IAV replication and IAV-induced inflammatory cytokine production (Figure 7).

PPARα/γ-AMPK Pathway and Fatty Acid Metabolism
Might Play Important Roles in the Inhibitory Effect of Emodin on IAV Infection and Influenzal Pulmonitis In Vivo. To further determine the importance of the PPARα/γ-AMPK pathway and fatty acid metabolism in the anti-IAV activity of emodin, an in vivo test was performed. As shown in Figure 8, comparing with the only IAV-infected control (IAV), emodin treatment (IAV+EMO) could significantly improve the average survival time of infected mice and reduce lung index, lung cytokines, and pulmonary viral load. Furthermore, in the antagonism tests, comparing with the IAV+EMO group, the inhibitors of PPARα (MK886) and AMPK (CC) could significantly counteract the effects of emodin. PPARγ inhibitor GW9662 could partially counteract the effects of emodin. ETO and palmitate could counteract the effects of emodin on average survival time, lung index, and production of lung cytokines, but not significantly on IAV replication. Additionally, emodin could significantly improve IAV-induced histopathological changes, whereas MK886, CC, GW9662, ETO, and PA could partially counteract the inhibition of emodin on IAV-induced histopathological changes ( Figure 9).
Finally, after IAV infection, emodin also significantly increased the lung mRNA expression of PPARα, PPARγ, AMPK, FABP5, CPT1A, CPTII, ACOX1, and MLYCD and  Figure 6: Influence of emodin on FAS activity, free fatty acid, and cellular ATP levels. The treatments were the same as mentioned in Figure 2. After 48 h, relative FAS activity was measured spectrophotometically by monitoring the oxidation of nicotinamide adenine dinucleotide phosphate at 340 nm (a). The levels of free fatty acid and ATP in A549 were also measured by the commercial kits (b, c). Values are presented as the mean ± SD of three independent experiments. * P < 0:05 vs. the normal group; # P < 0:05 vs. the only IAVinfected group. 9 BioMed Research International the phosphorylation levels of AMPK and ACC and decreased the mRNA expression of SREBP-1c, ACC, and FAS, but not significantly for CD36 ( Figure 10).

Discussion
In the past more than ten years, we have always engaged in the screening and classification of TCMs and their active compounds by different cell-based luciferase bioassays according to different pathogenic mechanisms of IAV. Recently, we have focused on the PPARα/γ-AMPK pathway and performed a cell-based PPRE-driven luciferase bioassay and found that several TCMs have high activity. After further research, we find that emodin is very effective in attenuating IAV replication, which promotes us to further explore the effects of emodin analogs on IAV infection. In this study, we find that several emodin analogs have better anti-IAV activity than emodin does and emodin possesses broad-spectrum anti-IAV effect, both of which suggest that emodin and its analogs are a group of promising anti-IAV drug precursors.

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To research the synergistic effect of emodin and its analogs with other TCMs and their active compounds, it is necessary to determine the pharmacological mechanism of emodin and its analogs. In the present study, we researched the pharmacological mechanism of emodin and found that emodin can significantly increase the expressions of PPARα, PPARγ, and AMPK and upregulate the phosphorylation and enzymatic activity of AMPK in A549 cells, no matter IAV infection or not, while treatments with inhibitors or transfections of siRNAs for PPARα, PPARγ, and AMPKα1 can significantly impair the inhibitory action of emodin on IAV replication and IAV-induced cell injury, indicating that the PPARα/γ-AMPK pathway involves in the inhibition of emodin on IAV replication and pathogenic mechanism. In fact, there are many reports about emodin activating the PPARα/γ-AMPK pathway [22].
Moreover, AMPK is a key cellular energy sensor, involved in lipid homeostasis and ATP balance regulation, and most PPAR agonists exert their physiological effects through activating AMPK. The PPARα/γ-AMPK pathway also is an important anti-inflammatory pathway. Activation of PPARα can inhibit NF-κB by enhancing the expression of IκBα or directly binding to RelA/p65 protein [23]. Activation of PPARγ can inhibit the expressions of TLR4 and NADPH oxidase p47phox [24] and antagonize the inflammatory pathways such as NF-κB, AP1, and STAT in respiratory virus infections [25]. AMPK activa-tion can inhibit oxidative stress and airway inflammation in mice [26]. In our present study, treatments with inhibitors or transfections of siRNAs for PPARα, PPARγ, and AMPKα1 can also significantly impair the inhibitory effect of emodin on IAV-induced production of IL-1β, IL-6, IL-8, TNFα, and IP-10.
Fatty acid oxidation is very important for cell proliferation and functional homeostasis, because the carbons of fatty acid, which substantially replenish the Krebs cycle, are incorporated into aspartate (a nucleotide precursor) and uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and finally incorporated into DNA.  i., respectively. The survival rates were observed for 14 days. The pulmonary cytokine and viral load were determined by ELISA and TCID50 assays, respectively. The lung index was evaluated by determining the percent of lung wet weight (g) to body weight (g) (lung index = lung wet weight ðgÞ ÷ body weight ðgÞ × 100%). * P < 0:05 vs. the IAV-infected group; # P < 0:05 vs. the IAV+emodin group.

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Reduction of fatty acid oxidation will deplete cell stores of deoxyribonucleoside triphosphates (NTPs) and further impair the de novo nucleotide synthesis for DNA replication [29]. In mitochondrial fatty acid oxidation (β-oxidation), CPT1 is the key rate-limiting enzyme that mediates the transportation of fatty acids across the mitochondrial membrane into the matrix [30]. CPT1A overexpression increases long-chain fatty acid oxidation; CPT1A inhibitor etomoxir significantly decreased intracellular ATP levels [31]. Peroxisomes are indispensable for α-oxidation of branch chain fatty acids and β-oxidation of very long-chain fatty acids (>22 carbons; VLCFA). VLCFA are first oxidized by peroxisomal β-oxidation rate-limiting enzyme ACOX1 [32].
As we expected, emodin could significantly increase the expression of fatty acid oxidation genes, such as CPT1A, CPTII, ACOX1, and MLYCD, and significantly increase the ATP level in A549 cells, no matter IAV-infected or not. But in the antagonism assay, our results have showed that CPT1 inhibitor ETO and 3-ketoacyl coenzyme A thiolase inhibitor TMZ cannot reverse the inhibition of emodin on IAV replication but further reduces IAV replication. A further assay has showed that ETO and TMZ per se can significantly inhibit IAV replication. These results might suggest that β-oxidation might also be needed for IAV replication, which we speculate might be due to the fact that fatty acid oxidation is important for the de novo nucleotide synthesis of DNA and is a major source of energy for both cell survival and viral replication. This speculation needs further study to prove.
The de novo fatty acid biosynthesis plays an important role in viral replication. The de novo fatty acid biosynthesis is required for the formation of viral envelopment or lipid modification of viral proteins in the replication of many viruses [36]. ACC is the first rate-limiting enzyme in the de novo fatty acid biosynthesis. FAS is the important enzyme that catalyzes the reaction of malonyl-CoA with acetyl-CoA, ultimately generating the 16-carbon fatty acid palmitate. Palmitate is further utilized for the synthesis of more complex glycerophospholipids, sphingolipids, and cholesterol. IAV infection can induce de novo fatty acid biosynthesis and cholesterol synthesis [11]. ACC inhibitor TOFA and FAS inhibitor C75 significantly inhibit IAV replication [12].
The de novo fatty acid biosynthesis is also regulated by AMPK; AMPK is a direct upstream kinase that suppresses the cleavage and nuclear translocation of sterol-regulatory element binding protein (SREBP) 1. SREBP is a key lipogenic transcription factor that regulates the de novo fatty acid biosynthesis by activating genes involved in fatty acid synthesis (such as ACC and FAS) and triglyceride synthesis (such as stearoyl CoA desaturase 1, SCD1) [37]. Activation of AMPK can inhibit the replication of coxsackievirus B3 by inhibiting fatty acid biosynthesis and cellular lipid accumulation. This restriction can be bypassed by treatment with the exogenous palmitate and siRNA AMPK [38].
In the present experiment, emodin can significantly decrease the expression of genes involved in fatty acid biosynthesis (such as SREBP-1c, ACC, and FAS), inhibit FAS activity, and reduce the level of free fatty acid in A549 cells. Exogenous palmitate can significantly impair the  Figure 9: Influence of emodin on the histopathological changes after IAV infection. Mice were treated as mentioned in Figure 8. On day 6 p.i., six mice from each group were sacrificed. The right lungs were used in the haematoxylin and eosin (H&E) staining assay. The magnification was 200x. The evaluation of histopathological scores was carried out in a double-blind trial. Data shown were the mean ± SD. * P < 0:05 vs. the only IAV-infected control; # P < 0:05 vs. the IAV+emodin group. 14 BioMed Research International inhibitory action of emodin on IAV replication and the production of inflammatory cytokines. In addition, there are some reports about emodin inhibiting fatty acid biosynthesis [39]. Finally, we have performed an in vivo test and have found that emodin treatment can significantly improve the average survival time and reduce lung index, lung cytokines, pulmonary viral load, and histopathological changes, while the inhibitors MK886, GW9662, CC, and ETO as well as palmitate can partially counteract the inhibitory effect of emodin. Emodin also can significantly increase the lung mRNA expression of PPARα, PPARγ, AMPK, FABP5, CPT1A, CPTII, ACOX1, and MLYCD; decrease the mRNA expression of SREBP-1c, ACC, and FAS; and increase the phosphorylation levels of AMPK and ACC. Similar research has reported that emodin significantly ameliorates LPS-induced ALI/ARDS in mice by suppressing LPS-induced downregulation of PPARγ and upregulation of NF-κB p65 [40].

Conclusion
In the present study, we have performed a cell-based PPREdriven luciferase bioassay and found that several emodin analogs possess better anti-IAV activity than emodin does, and emodin possesses broad-spectrum anti-IAV activity. Mice were treated as mentioned in Figure 8. On day 6 p.i., lung tissues were collected and homogenated. The mRNA levels of target genes were measured by qRT-PCR (a, b). The phosphorylation levels of AMPK and ACC were determined by an ELISA assay (c). Data shown were the mean ± SD. n = 6. * P < 0:05 vs. the only IAV-infected control.