Picroside II Improves Severe Acute Pancreatitis-Induced Intestinal Barrier Injury by Inactivating Oxidative and Inflammatory TLR4-Dependent PI3K/AKT/NF-κB Signaling and Improving Gut Microbiota

Background Picroside II exerts anti-inflammatory and antidiarrheal effects for treating the diseases associated with oxidative injury. However, its function on pancreatitis-induced intestinal barrier injury remains unclear. Hypothesis/Purpose. We hypothesized that picroside II will have protective effects against pancreatitis-induced intestinal barrier injury by affecting oxidative and inflammatory signaling (Toll-like receptor 4- (TLR4-) dependent phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), and nuclear factor kappa B (NF-κB)). Study Design and Methods. A Sprague-Dawley (SD) rat model with severe acute pancreatitis (SAP) was induced via the injection of sodium taurocholate (4% wt/vol; 1 mL/kg). All rats were divided into 3 groups: sham (CG), SAP-induced intestinal barrier injury (MG), and picroside II (PG) groups. Intestinal barrier injury was assessed by scanning electron microscopy (SEM), hematoxylin and eosin staining, and pathological scores. We measured the levels of pancreatitis biomarkers (amylase and lipase), oxidative and inflammatory signaling (TLR4-dependent PI3K/AKT/NF-κB), oxidative stress marker (superoxidase dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), and malondialdehyde), and inflammatory markers (tumor necrosis factor α (TNFα), interleukin- (IL-) 1, IL-6, and IL-10) in serum and/or gut tissues. Gut microbiota composition in feces was measured by using 16S rRNA sequencing. Results SEM showed that intestinal barrier injury was caused with the loss of intestinal villi and mitochondria destruction, and pathological scores were increased in the MG group. The levels of amylase, lipase, malondialdehyde, TNFα, IL-1, IL-6, TLR4, PI3K, AKT, and NF-κB were increased, and the levels of SOD, GPx, CAT, and IL-10 was reduced in the MG group when compared with CG group (P < 0.05). Picroside II treatment inhibited the symptoms in the MG group and showed antioxidant and anti-inflammatory activities. The serum levels of picroside II had strong correlation with the levels of inflammatory and oxidative stress biomarkers (P < 0.05). Picroside II treatment increased the proportion of Lactobacillus and Prevotella and decreased the proportion of Helicobacter and Escherichia_Shigella in the model. Conclusions Picroside II improved the SAP-induced intestinal barrier injury in the rat model by inactivating oxidant and inflammatory signaling and improving gut microbiota.


Introduction
Severe acute pancreatitis (SAP) is associated with multiple organ failure and systemic inflammatory responses with a high fatality rate of up to 15-20% [1]. Although much progression has been made in diagnostic strategies [2] and therapeutic methods [3] for SAP in recent, an effective therapeutic drug is still unavailable. Picroside II is an active constituent extracted from herbs [4,5] and has long been used as traditional Chinese medicine for treating the diseases associated with oxidative injury and acute inflammation [5][6][7]. However, the role and underlying pharmacological mechanisms of picroside II in SAP are largely unknown.
Oxidative stress and the activation of inflammatory responses have been regarded to play important roles in SAP progression [8,9]. Our previous work showed that picroside II ameliorated SAP progression by increasing antioxidant and anti-inflammatory activities of SAP-induced intestinal barrier injuries via nuclear factor kappaB-(NF-κB-) dependent autophagy [10]. Actually, intestinal barrier plays an important role in the prevention of SAP risk [11], and SAP incidence can induce intestinal barrier injury [12]. The change of gut microbiota may lead to barrier failure, which is a key event contributing to the severity of gut injury [13,14]. Toll-like receptor 4-(TLR4-) dependent phosphatidylinositol 3-kinase (PI3K)/Protein kinase B (AKT)/NF-κB signaling is closely associated with oxidative stress and inflammatory responses [15,16]. However, whether picroside II exerts its function on SAP-induced intestinal barrier injury or affects TLR4-dependent PI3K/AKT/NF-κB signaling remains unknown. Therefore, in this study, we aimed to explore the related molecular mechanism for the protective effects of picroside II in the model with SAP-induced intestinal barrier injury.

Establishment of SAP-Induced Intestinal Barrier Injury.
All animal-related procedures were approved by the Institutional Animal Care and Use Committee of Jinzhou Medical University (Jinzhou, China). Ninety male Sprague-Dawley (SD) rats (8 weeks old; weighing 200-220 g) were purchased from the animal center of Jinzhou Medical University (Jinzhou, China). The rats were maintained on a 12-hourlight/12-hour-dark cycle at 22°C, given a standard laboratory diet (Tecklab, Winfield, IA, USA) and water ad libitum and allowed to acclimatize for a week. SAP was established via the injection of 0.2 mL of 5% sodium taurocholate into the common biliopancreatic duct [17]. Meanwhile, in the sham group, the rats were injected with the same volume of saline solution.

Measurement of Serum
Amylase and Lipase. One mL blood was withdrawn from the tail of each rat after 3-, 6-, and 24-day picroside II administration. Serum was prepared via centrifugation at 1,000 × g for 10 min and stored at −20°C for ELISA, amylase, and lipase measurement. Amylase assay kit was purchased from Abcam (ab102523, Cambridge, MA, USA), and Lipase ELISA kit was purchased from Life Science Inc. (Wuhan, China). Their activities were measured on an automatic biochemical analyzer (Dimension, Schererville, IN, USA).

Scanning Electron Microscopy Observation of Intestinal
Barrier. For SEM processing, about 5 mm 2 of gut mucosa were cut from each rat after 24-day picroside II administration and fixed with 1% osmium tetroxide for 2 h at 4°C. The tissues were rinsed, dehydrated in ethyl alcohol, dried with carbon dioxide, covered with gold, and examined under SEM JSM-6610lv (Jeol, Japan) with an INCA SDD X-MAX energy dispersive microanalyzer.

2.7.
Histological Analysis of Small Intestine Tissues. Pancreatic tissues were extracted after 3-, 6-, and 24-day picroside II administration via intraperitoneal injection of phenobarbital sodium (50 mg/kg) (n = 10 for each group at each time). Some small intestine tissues were fixed in 4% paraformaldehyde and embedded in paraffin and remaining tissues were stored in -80°C. The embedded pancreatic tissues were cut into 2-3 μm slices and stained with hematoxylin and eosin (H&E). Pancreatic edema and the numbers of inflammatory cell infiltration, bleeding, and necrotic cells were calculated. The severity of pancreatic tissue damage was assessed by using pathological score = edema score + necrosis score + inflammatory cellular infiltration score + bleeding score. Five slices were evaluated in each group.
2.8. Immunohistochemistry Analysis. Immunohistochemistry analysis was conducted to evaluate the expression of TLR4, PI3K, AKT, and NF-κB. The paraffin-embedded tissue sections were deparaffinized and treated with hydrogen peroxide (3 m/v) for 15 min to remove endogenous peroxidase. Antigen retrieval was performed by blocking the samples in goat serum for 10 min at 22°C. The following antibodies were added and incubated 12 h at 4°C, including anti-TLR4 antibody (ab13867, 1 : 500), anti-p-PI3K p85 antibody (Abcam, ab86714, 1 : 500), anti-p-AKT antibody (ab38449, 1 : 500), 2 Oxidative Medicine and Cellular Longevity and/or anti-p-NF-κB antibody (ab86299) from Abcam (Cambridge, MA, USA). A biotin-labeled goat anti-rabbit IgG secondary (1 : 1000) was added followed by incubation at 37°C for 10 min. The slides were then incubated at 37°C for 10 min with peroxidase-conjugated streptavidin (Sigma, S5512). The sections were stained with 3,3 ′ -diaminobenzidine (DAB, sigma) and counterstained with hematoxylin (Sigma). Color separation was conducted by using 2% hydrochloride and alcohol, followed by 15 min washing. Each sample was observed in five, and target protein signals were stained with brown. The positive rates were calculated as the number of positive cells/the number of total cells by using an image analyzer (Image-Pro Plus 5.1, MediaCybernetics, MD, USA).
2.10. Western Blot Analysis. Ten mg pancreatic tissue was ground in liquid nitrogen, and total protein was extracted by using RIPA lysis (CST, Danvers, MA, USA). Protein concentration was quantified using the BCA kit (TaKaRa, Dalian, China). HRP-conjugated goat anti-rabbit IgG H&L (ab6721) secondary antibodies were from Abcam (Abcam, San Francisco, CA, USA). The proteins were separated by SDS-PAGE and transferred to the PVDF membrane in the transfer buffer at 100 V for 2-3 h. The membrane was blocked for 1 hour at ambient room temperature in 10% nonfat milk, probed with antibodies against the above primary antibodies for 2 hours at 37°C, rinsed four times with PBTB, incubated 2 hours at 37°C in secondary antibodies, and washed extensively in PBS. Images were acquired on an Odyssey CLx infrared scanner (Li-Cor-Nebraska USA). Relative protein levels were calculated by using internal reference β-actin.
2.11. Gut Microbiota Analysis. About 10 mg fresh feces were obtained from each rat after 3-, 6-, and 24-day picroside II administration. The genome of gut microbiota was isolated using a FastDNA Spin Kit (Qbiogen, Carlsbad, CA, USA). 16S rRNA was amplified by PCR using forward primer 5 ′ -GAGAGTTTGATCCTGGCTCAG-3 ′ and the reverse primer 5 ′ -GGTTACCTTGTTACGACTT-3 ′ . Gut microbiota was analyzed by using 16S rRNA sequencing. Heat map and taxon relative abundance bar diagram was created by using custom R scripts and ggplot2.
2.12. Statistical Analyses. Data are presented as the mean values ± standard deviation (S.D.) and analyzed using the SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA). Student's t-test and one-way analysis of variance (ANOVA) with post hoc Tukey's tests were used to evaluate the variables between groups. The statistical difference was significant if the value of P < 0:05.

Picroside II Treatment Reduced the Activities of SAP
Biomarkers. Amylase and lipase are the potential biomarkers of pancreatitis [18]. After the establishment of SAP, the activities of serum amylase (Figure 1(a)) and lipase (Figure 1(b)) in the MG group were higher than those in the CG group (P < 0:05). Picroside II treatment reduced the activities of serum amylase (Figure 1(a)) and lipase (Figure 1(b)) in the PG group when compared with those in the MG group after 3-, 6-, and 24-day intervention (P < 0:05).  (Figure 4(a)), IL-1 (Figure 4(b)), and IL-6 ( Figure 4(c)) were increased, while the serum level of IL-10 ( Figure 4(d)) was reduced in the MG group when compared with the CG group (P < 0:05). Picroside II treatment reduced the serum levels of TNFα (Figure 4(a)), IL-1 (Figure 4        Oxidative Medicine and Cellular Longevity 3.9. Picroside II Treatment Improved Gut Microbiota in the SAP-Induced Intestinal Barrier Injury. Bar plot showed that the proportion of Lactobacillus were decreased in the MG group, and Prevotella was decreased except of after 24-day picroside II intervention when compared with the CG group (Figure 9(a)). Picroside II treatment increased the proportion of Lactobacillus and Prevotella and decreased the proportion of Helicobacter and Escherichia_Shigella in the model (Figure 9(a)). The proportional change of major gut microbiota was provided as supplementary Table S1. Heat map also showed that the levels of Lactobacillus were decreased, and Prevotella was decreased except of after 24-day picroside II intervention in the MG group when compared with the CG group (Figure 9(b)). Picroside II treatment increased the levels of Lactobacillus and Prevotella and decreased the proportion of Helicobacter and Escherichia_Shigella in the model (Figure 9(b)). The results suggest that picroside II treatment improved gut microbiota in the SAP-induced intestinal barrier injury.

Discussion
In the present experiment, the administration of sodium taurocholate was conducted to establish SAP-induced intestinal barrier injury, and pathological changes were found in the pancreatic tissues of MG group when compared with the CG group ( Figures 5 and 6). The activities of SAP biomarker (serum amylase and lipase) were also increased ( Figure 1). These results suggest that sodium taurocholate could induce SAP in rats, which had higher pathological scores in the small intestine ( Figures 5 and 6). Meanwhile, oxidative stress and the inflammatory responses were also increased ( Figures 3 and 4), suggesting that sodium taurocholate is an effective agent to induce SAP in the rat model and also used in other SAP studies [19,20]. Picroside II treatment also affected the expression levels of TLR4-dependent phosphorylated PI3K/AKT/NF-κB ( Figure 8) but not the relative mRNA levels of PI3K/AKT/NF-κB. The results suggest that picroside II may also exert its function by affecting phosphorylated situation of PI3K/AKT/NF-κB via TLR4. The results were partially consistent with previous report that hydrogen sulfide mitigates SAP via PI3K/AKT/NF-κB pathway [21]. The result also suggests that picroside II may exert its antioxidant and anti-inflammatory properties in SAP-induced intestinal barrier injury by inactivating MAPK/NF-kappaB signaling. Picroside II may be an effective compound to prevent SAP development with fewer side effects without toxicity as natural products [22].
The improvement of antioxidant and anti-inflammatory properties is the potential approaches for preventing SAP progression [23,24]. In the present work, picroside II protected rats against SAP development by increasing antioxidant and anti-inflammatory capacities (Figures 3 and 4). Picroside II may be affective to increase the antioxidant and anti-inflammatory activities in the prevention of SAP progression. SAP-induced intestinal barrier injury may cause the change of gut microbiota ( Figure 9) and result in Relative protein level of p-NF-B    (b) Figure 9: The composition of gut microbiota among different groups. (a) The proportion of gut microbiota. (b) Heat map analysis of gut microbiota changes from different treatments. G1-3 stands for the CG, MG, and PG groups at 3 d, respectively; H1-3 stands for the CG, MG, and PG groups at 6 d, respectively; I1-3 stands for the CG, MG, and PG groups at 24 d, respectively. treatment increased the proportion of Lactobacillus and Prevotella and decreased the proportion of Helicobacter and Escherichia_Shigella. Lactobacillus species exerts protective effects on intestinal integrity and immune responses of the animal infected with Clostridium [25]. The improvement of anti-inflammatory status has been reported to be followed by the increase in the abundance of Prevotella [26]. Intestinal barrier injury is closely associated with numerous factors, such as bacterial infection, inflammation, and mechanical damage; all of which can be caused by Helicobacter and Escherichia_Shigella infection [27,28]. Thus, picroside II treatment may ameliorate intestinal barrier injury by improving the proportion of gut microbiota.

Data Availability
All data related to this paper may also be requested from the corresponding authors (with a lead contact at the Email: liubaoh627@163.com).

Conflicts of Interest
The authors declare that they have no conflicts of interest.