a b s t r a c t

Background: Shenfu injection (SFI) promotes tissue microcirculation and Oxygen metabolism. We aimed to assess its effects on intestinal epithelial damage in septic rats.

Methods: Fifty Sprague-Dawley rats were randomly divided into sham operation (Sham), sepsis (cecal ligation and puncture [CLP]), and SFI (low-dose, middle-dose, high-dose) groups (n = 10). For Sham animals, the abdom- inal cavity was opened and closed. For other groups, severe sepsis was induced by CLP. After surgery, saline (Sham and CLP rats) and SFI (treatment groups) were administered intraperitoneally. Samples were collected 12 hours after injection. Serum tumor necrosis factor ?, diamineoxidase, and D-lactate levels and ileal mucosal damage and ultrastructural change, as well as protein and Messenger RNA expression of tight junction markers, including Claudin-3 and zonula occludens protein-1 in ileal mucosa’s epithelial cells, were assessed. All Animal experiments were carried out under aseptic conditions.

Results: Compared with Sham animals, serum tumor necrosis factor ?, DAO, and D-lactic acid levels in CLP ani- mals were significantly higher; the ileal mucosal damage was more severe; and the expression levels of tight junction markers were significantly decreased. These indexes were significantly improved in SFI groups, in a concentration-dependent manner, compared with CLP rats. Sham animals displayed orderly arranged ileal mu- cosal villi, continuous tight junctions between epithelial cells, intact organelles, and microvilli. Compared with CLP animals (with obvious damage in these structures), an overt improvement was observed in SFI groups, espe- cially in the high-dose SFI group, with tight junctions clearly visible between epithelial cells.

Conclusions: Shenfu injection significantly alleviates intestinal epithelial damage in septic rats, in a dose- dependent manner.

(C) 2015

Introduction

Severe sepsis is treated by early fluid resuscitation and appropriate an- tibiotics, although a high mortality rate of 30% to 50% is still observed [1]. Before 1986, the gastrointestinal (GI) tract was believed to play a passive role in the pathophysiology of sepsis. In 1986, Carrico et al [2] first pro- posed the GI tract to be the driving force behind multiple-organ dysfunc- tion syndrome in sepsis. In 1987, the concepts of Bacterial translocation from the gut and gut-origin sepsis were proposed by Border et al [3]. To date, many studies have demonstrated the GI tract’s significance in the initiation and progression of systemic inflammatory response syndrome, sepsis, and multiple-organ dysfunction syndrome. Researchers have also demonstrated the importance of the Intestinal mucosa‘s mechanical bar- rier in preventing bacterial translocation from the gut, with the intercellu- lar tight junctions playing a critical role [4]. Tight junctions consist of the tight junction proteins occludin, claudins, junctional adhesion molecules, and tricellulin. These proteins form a tight junction complex with junc- tional complex proteins, such as zonula occludens protein-1 (ZO-1), via

? Source of support: This work is supported by Prevention and Control of Major Dis- eases, Zhejiang Chinese Medicine Research Program (No. 2012ZGG001).

?? Conflict of interest: The authors declare that they have no conflict of interest.

* Corresponding author. Tel.: +86 13958095268; fax: +86 21 64085875.

E-mail address: jiangrl0420@sina.com (R. Jiang).

the cell cytoskeleton system [5]. Tight junction was demonstrated to be highly correlated with intestinal permeability [6].

However, the protective effect of Shenfu injection (SFI) on tight junctions of intestinal mucosa’s epithelial cells is not well understood, and the underlying mechanisms need to be elucidated. In this study, we hypothesized that SFI could significantly alleviate intestinal epithe- lial damage in septic rats. Therefore, a rat model of severe sepsis was established by cecal ligation and puncture (CLP). The tight junction changes in intestinal mucosa’s epithelial cells and the protective effects of different SFI doses were examined. In addition, the mechanisms by which SFI protects the intestinal mucosa’s mechanical barrier were ex- amined. Our data reveal SFI’s preventive effects on the pathophysiolog- ical processes of sepsis, indicating that SFI can further improve the success rate of resuscitation in patients with severe sepsis.

Materials and methods

Animal grouping

The study protocol was approved by the hospital’ Animal Research Ethics Committee. A total of 50 clean-grade healthy male Sprague- Dawley rats weighing 200 +- 20 g were provided by the experimental

http://dx.doi.org/10.1016/j.ajem.2015.08.001

0735-6757/(C) 2015

Western blot analysis of tight junc”>animal center of Zhejiang Chinese Medical University, China. Animals were housed for 2 weeks under normal conditions in the experimental animal center of Zhejiang Chinese Medical University at 20?C +- 1?C and 50% to 60% humidity, under 12:12-hour light/dark cycle, with a ventilation rate of 8 to 15 times/h. Using a random number table, rats were subdivided into 5 groups (n = 10): sham operation (Sham), sepsis (CLP), and SFI (low-dose, LSF; middle-dose, MSF; high-dose, HSF) groups.

Animal model establishment and treatment

A rat model of severe sepsis was induced by CLP [7]. Briefly, all rats were deprived of food, but with free access to water for 12 hours prior to surgery. Anesthesia was carried out with an intramus- cular injection of 5% ketamine (0.2 mL/100 g body weight). After fix- ation on the operating panel, rats were submitted to abdominal hair removal and skin disinfection, and a ventral Midline incision (1.5 cm) was made. For Sham rats, the abdominal cavity was opened and closed immediately, but was not ligated or perforated. For rats of the remaining 4 groups, the abdominal cavity was opened to find the cecum, whose mesentery was carefully dissected; then, the cecum root was ligated, avoiding damage to the ileum and mesenter- ic vessels. Punctures were made through the cecum by perforating at 3 locations using a 21-gauge needle. The cecum was gently com- pressed until fecal pellets were extruded. The bowel was then returned to the abdomen and the incision closed. At the end of the operation, all rats were resuscitated immediately with normal saline (5 mL/100 g body weight) subcutaneously. Ten minutes after opera- tion, Sham and CLP animals received 20 mL/kg of normal saline; the rats of the LSF, MSF, and HSF groups received 5 mL/kg SFI plus 15 mL/ kg of normal saline, 10 mL/kg SFI plus 10 mL/kg normal saline, and 20 mL/kg SFI, respectively, by tail vein injection. Afterward, the rats were placed in cages at a constant temperature (22?C) and allowed free access to food and water. The animals were constantly observed until they recovered from anesthesia. Dead animal number, animal vigor, hair and stool characteristics of survival rats, and number of open abdominal cavities were evaluated every 2 hours.

Animal model verification and specimen collection

Twelve hours after operation, survival rats were anesthetized with 5% ketamine. To verify whether the animal model was success- fully established, rats in Sham and CLP groups were examined. Heart rate (HR) was measured as follows: needle electrodes were inserted into both upper extremities and Left lower extremity and connected to the RM-6280 Physiologic Recording System (Junyue electrical equipment factory, Dongguan, China). Rectal temperature (T) was monitored using a thermometer. Heart blood samples were collected as follows: hair removal and skin disinfection were performed in the area under the xiphoid; then, a needle attached to a 5-mL syringe was inserted upward into the heart at 30?, and 2 mL blood was drawn from heart into a blood culture flask. The abdominal cavity was opened and swabbed with a sterile swab stick to collect 1 mL peritoneal exudates. The peritoneal swabs and blood culture speci- mens were immediately sent to the Clinical Pathogenic Microbiolog- ical Laboratory of the First Affiliated Hospital of Zhejiang Chinese Medicine University for Bacterial culture [8]. For all rats, blood sam- ples (2 mL) were collected from the abdominal aorta; a 2-cm section of terminal ileum was extracted for further analysis; a portion of blood specimen was immediately sent to the Clinical Laboratory of the above mentioned hospital for white blood cell (WBC) amounts, serum aminotransferase (ALT), aspartate transferase (AST), Blood urea nitrogen , and Creatinine levels, and creatine kinase iso- enzyme MB (CK-MB) activity.

Detection of serum tumor necrosis factor ?, diamineoxidase, and D-lactic acid levels

Serum tumor necrosis factor ? (TNF-?), diamineoxidase (DAO), and D-lactic acid concentrations were measured using enzyme-linked im- munosorbent assay kits following the manufacturer’s instructions (Shanghai Xitang Biotechnology, LTD, Shanghai, China).

Ileal mucosal damage index

Ileum tissue sections (3-5 um) were stained with hematoxylin and eosin (HE) and observed under a XZT-302 microscope (Shanghai Yuguang Detection Equipment Co, Ltd, Shanghai, China) at x100 magni- fication. Histologic evaluation was performed according to the Chiu scor- ing method [9].

Ultrastructure of ileal mucosal epithelium

Ileum tissue (1 mm³) fixed by glutaraldehyde was obtained and fixed again with 1% osmic acid at 4?C for 1 hour. Then, samples were dehydrated twice with ethanol gradient (50%, 70%, 80%, 90%, and 100%) and acetone. After dehydration, the tissue was embedded in acetone-Epon812 and penetrated. The resulting tissue block was placed into a capsule with diallyl phthalate, which was allowed to polymerize for 72 hours (24 hours each at 35?C, 45?C, and 60?C). After polymeriza- tion, the tissue block was trimmed, and ultramicrotomy was performed. The thin sections were double stained with lead citrate and uranyl ace- tate, and the ultrastructure of ileal mucosa’s epithelium was observed on a JEM-12003X transmission electron microscope (JEM, Akishima, Japan).

Western blot analysis of tight junction protein expression

Twenty milligrams of ileal tissue was minced, resuspended in 200 uL RIPA buffer, and homogenized. Protein concentration was determined by the bicinchoninic acid method. Thirty micrograms of total protein ex- tract was subjected to sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis and transferred onto a membrane. After blocking with 5% nonfat milk for 2 hours followed by 3 washes for 30 minutes in Tween-tris buffered saline buffer (10 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 0.05% Tween-20), the membrane was incubated with rab- bit anti-Claudin-3 (1:2000; Abcam, Cambridge, UK) and anti-ZO-1 (1:2000; Abcam) polyclonal antibodies, overnight at 4?C. The mem- brane was washed 3 times and incubated with horseradish peroxidase-labeled goat antirabbit secondary antibody for 2 hours. Sig- nals were detected on a Bio-Rad (Hercules, CA) gel imaging system, and protein bands were semiquantified with the Quantity One software (Bio-Rad Laboratories, Hercules, CA). The relative tight junction protein level was derived as an optical density value of the tight junction pro- tein/optical density value of ?-actin from the same sample.

Determination of messenger RNA expression of tight junction genes

The messenger RNA (mRNA) expression of tight junction genes in ileal mucosa’s epithelial cells was assessed by reverse transcriptase po- lymerase chain reaction (PCR). Total RNA was extracted using TRIzol re- agent (Life Technologies, Waltham, MA). Then, first-strand cDNA was synthesized with the Super RT cDNA Kit (Kangwei Shiji Biotechnology, LTD, Beijing, China) following the manufacturer’s instructions. Real- time PCR was performed on an ABI 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA), with initial denaturation at 95?C for 10 minutes, followed by 35 to 40 cycles of 95?C for 15 seconds and 60?C for 1 minute. The primers used are as follows: 5?- CATCGCAGCTACTTGCCAGT-3? (forward) and 5?-TTTTTTTTTTTTTTTTGC

AAAAACGA-3? (reverse) for Claudin-3; 5?-CCATCTTTGGACCGATTGC TG-3? (forward) and 5?-TAATGCCCGAGCTCCGATG-3? (reverse) for ZO-

1; 5?-GGCACCACACTTTCTACAATGA-3? (forward) and 5?-TCTCTTTAAT

GTCACGCACGAT-3? (reverse) for actin. Data were analyzed by the com- parative CT method (-2^??Ct) with actin as internal control.

Statistical analysis

Statistical analysis was performed using the SPSS 17.0 software (SPSS, Chicago, IL). Data were expressed as mean +- SD (x+- s) for mea- surement data following a Gaussian distribution. Multiple comparisons were carried out by analysis of variance. The least significant difference test was used for pairwise comparisons; log-rank test was used to com- pare the survival distributions between groups. P b .05 was considered statistically significant.

Results

The sepsis model was successfully established

Twelve hours after the operation, bacterial cultures of blood and peritoneal exudates were performed. No pathogenic bacteria were found in the Sham group; in the CLP group, however, blood cultures were positive for Enterococcus faecalis, Escherichia coli, and Proteus mirabilis, and similar results were obtained for peritoneal exudate cul- tures, which were positive for Bacillus proteus vulgaris, P mirabilis, E faecalis, and E coli. As shown in Table 1, HR, T, and blood WBC of CLP an- imals were significantly higher compared with values obtained for the Sham group (P b .05). In addition, the serum ALT, AST, BUN, and Cr levels, as well as CK-MB activity, were all significantly higher (P b .05).

Shenfu injection improves the general condition of rats

During the experimental sessions, no rats died in the Sham and MSF groups; 1 rat died in each CLP, LSF, and HSF groups; this corresponded to mortality rates of 0, 0, 10.0%, 10.0%, and 10.0%, respectively. There was no significant difference between groups (P N .05). Sham animals were fit and alert, and had bright glossy hair, normal stool, no fishy odor in the abdominal cavity, and no intestinal hyperemia or edema. The live rats of the CLP group seemed depressed, showing delayed response, poor appetite, loose stool without shape, lusterless hair, and bloody as- cites; they also had a strong fishy odor in the abdominal cavity, intesti- nal hyperemia and edema, and a dark-purple ligated cecum. The survival animals in the LSF, MSF, and HSF groups had acceptable fitness and showed a more flexible response, normal appetite, stool without shape, lackluster hair, and bloody ascites; in addition, they had a strong fishy odor in the abdominal cavity, intestinal hyperemia and edema, and a dark-purple distal end of the ligated cecum. There were no significant differences among SFI groups.

Shenfu injection has no adverse effects on organs

In order to assess the adverse effects of SFI, blood samples were col- lected 12 hours after the operation from rats of the LSF, MSF, and HSF groups. The following values were obtained for various parameters in the LSF group: ALT, 92.3 +- 3.4 U; AST, 99.5 +- 4.3 U; BUN, 10.4 +- 1.3

mmol/L; Cr, 113.9 +- 12.3 umol/L; and CK-MB, 38.3 +- 2.6 U/L. In the MSF group, similar values were obtained: ALT, 95.1 +- 4.5 U; AST,

94.7 +- 5.5 U; BUN, 11.8 +- 2.8 mmol/L; Cr, 114.5 +- 14.6 umol/L; and

CK-MB, 37.7 +- 3.0 U/L. These following values were recorded for the HSF group: ALT, 94.2 +- 3.7 U; AST, 96.3 +- 3.5 U; BUN, 11.3 +- 3.2

mmol/L; Cr, 116.3 +- 11.6umol/L; and CK-MB, 37.3 +- 1.7 U/L. There were no significant differences between LSF/MSF/HSF groups and the CLP group (P N .05).

Shenfu injection decreases serum TNF-?, DAO, and D-lactic acid levels in a dose-dependent manner

As shown in Table 2, serum TNF-?, DAO, and D-lactic acid levels in other groups were significantly higher (P b .05) compared with the Sham group. Interestingly, SFI treatment resulted in significantly de- creased serum TNF-?, DAO, and D-lactic acid levels compared with the CLP group (P b .05). These levels also significantly decreased with in- creasing SFI dosages (HSF b MSF b LSF, P b .05).

Shenfu injection improves the intestinal mucosal morphology

In the Sham group, the ileal mucosa’s villi showed an orderly ar- rangement. In the CLP group, these structures were obviously damaged: the epithelial cells at villus tips were wiped off; the subepithelial capil- laries exhibited congestion; the central lacteals were expanded; the lamina propria was exposed and disintegrated; and blood capillaries were bleeding, with ulcer formation and widened intracellular tight junctions. Compared with the CLP group, the morphologies of ileal mu- cosal villi and intestinal glands in the LSF, MSF, and HSF groups were im- proved to varying degrees, with significantly lower Chiu’s pathological scores (P b .05); compared with the MSF group, a more pronounced im- provement in ileal mucosal morphology was observed in the HSF group, with significantly lower Chiu’s pathological scores (P b .05; Table 3 and Fig. 1A-E). Electron micrographs (26,500x) showed ileal mucosal villi in an orderly arrangement for Sham animals, with continuous tight junc- tions between epithelial cells (Fig. 1F). In the CLP group, the ileal muco- sal microvilli were thinner and shorter, with some of them disrupted; tight junctions were discontinuous, and intercellular spaces were wid- ened (Fig. 1G). In the LSF, MSF, and HSF groups, the pathological changes were less pronounced: ileal mucosal microvilli were largely arranged in order and tight junctions were continuous, especially in the HSF group, where intercellular tight junctions were clearly visible (Fig. 1H-J).

Shenfu injection increases the expression of the tight junction proteins Claudin-3 and ZO-1

Compared with the Sham group, the tight junction protein (Claudin- 3 and ZO-1) levels in ileal tissues were significantly decreased in the CLP group (P b .05). Compared with the CLP group, Claudin-3 and ZO-1 ex- pression levels were significantly increased in the LSF, MSF, and HSF groups (P b .05). The expression levels of these tight junction proteins increased with SFI dosage, which indicates a concentration-dependent effect (Fig. 2).

Shenfu injection increases the mRNA levels of the tight junction proteins Claudin-3 and ZO-1

Compared with Sham rats (n = 10), Claudin-3 and ZO-1 mRNA levels in ileal tissues were significantly lower in all other 4 groups (P b

.05). Compared with the CLP group, Claudin-3 and ZO-1 gene

Table 1

Comparison of HR, T, blood WBC, ALT, AST, BUN, Cr, and CK-MB between Sham and CLP groups

Group	HR (beats/min)	T (?C)	WBC (x109)	ALT (U/L)	AST (U/L)	BUN (mmol/L)	Cr (umol/L)	CK-MB (U/L)
Sham (n = 10)	327.2 +- 35.6	37.3 +- 0.2	6.2 +- 1.5	34.9 +- 3.2	37.2 +- 1.5	7.3 +- 0.7	53.7 +- 4.5	19.1 +- 3.3
CLP (n = 9)	552.3 +- 27.4?	38.7 +- 0.2?	13.6 +- 1.1?	95.4 +- 2.5?	98.7 +- 3.2?	11.3 +- 1.3?	115.3 +- 11.5?	37.1 +- 2.1?

Data are mean +- SD. Sham, sham operation group; CLP, severe sepsis group.

* P b .05 vs Sham group.

Table 2

Serum TNF-?, DAO, and D-lactic acid levels in rats of different groups

Group	TNF-? (pg/mL)	DAO (U/L)	D-Lactic acid (mmol/L)
Sham (n = 10)	46.23 +- 7.76	583.42 +- 41.68	1.46 +- 0.08
CLP (n = 9)	124.40 +- 8.87?	1956.34 +- 51.76?	3.44 +- 0.14?
LSF (n = 9)	105.60 +- 2.44??	1795.79 +- 58.93??	2.83 +- 0.06??
MSF (n = 10)	90.21 +- 5.57?????	1171.88 +- 49.11?????	2.48 +- 0.09?????
HSF (n = 9)	61.50 +- 7.14?????+	915.01 +- 82.45?????+	2.45 +- 0.69?????+

Sham, sham operation group; CLP, severe sepsis group; LSF, low-dose SFI group; MSF, mid- dle-dose SFI group; HSF, high-dose SFI group.

* P b .05 vs sham.

?? P b .05 vs CLP.

??? P b .05 vs LSF.

⁺ P b .05 vs MSF.

expression was significantly increased in the LSF, MSF, and HSF groups (P b .05). The mRNA expression levels of these tight junction genes sig- nificantly (P b .05) increased with SFI dosage (HSF N MSF N LSF), indicat- ing a dose-dependent effect (Fig. 3).

Discussion

In this study, SFI improved the general conditions of sepsis rats; re- duced the sepsis-induced serum TNF-?, DAO, and D-lactic acid levels in a dose-dependent manner; improved the morphology of intestinal muco- sa; and increased the protein and mRNA expressions of the tight junction markers Claudin-3 and ZO-1, which are down-regulated by sepsis.

As shown above, HR, body temperature, and WBC count, indicators of inflammation, were significantly higher in the CLP group than in Sham animals. Liver and renal function indexes were evaluated, and ALT, AST, BUN, and Cr levels were significantly higher in the CLP group compared with the Sham group. In addition, bacteria were observed for both blood and the peritoneal exudate cultures in the CLP group. According to the criteria in the Guidelines for the Diagnosis and Treatment of Sepsis: 2012 [10], the severe sepsis model was thus established successfully.

The intestinal barrier consists of mechanical, immunologic, chemi- cal, and biological components, with the mechanical barrier being the most important; the latter is composed of mucosal epithelium, intercel- lular tight junctions, lamina propria, adherens junctions. and intestinal secretions. The integrity and regeneration capacity of the mucosal epi- thelium promote its barrier function [11], with tight junctions between cells playing the most important role [12]. An intact epithelium can ef- fectively prevent microorganisms from penetrating across mucosa and causing toxemia and bacteremia, and subsequently strong inflammato- ry response and multiple-organ damage.

Tumor necrosis factor ?, the main inflammatory mediator of sepsis, was shown to induce Caco-2 tight junction permeability, an effect mediated by activation of the ERK1/2 signaling pathway; in addition, in vivo intestinal perfusion studies revealed that the TNF-?-induced increase in mouse intes- tinal permeability requires ERK1/2-dependent activation of Elk-1 [13].

Increased Serum D-lactate levels and DAO activities were observed in rats with acute lung injury/acute respiratory distress syndrome [14].

Table 3

Comparison of the intestinal mucosal damage index of rats in different groups

Group Chiu’s pathological score

Sham (n = 10) 0.25 +- 0.46

CLP (n = 9) 4.00 +- 0.76?

LSF (n = 9) 2.13 +- 0.64??

MSF (n = 10) 2.00 +- 0.76?????

HSF (n = 9) 1.88 +- 0.64?????+

Sham, sham operation group; CLP, severe sepsis group; LSF, low-dose SFI group; MSF, mid- dle-dose SFI group; HSF, high-dose SFI group.

* P b .05 vs Sham.

?? P b .05 vs CLP.

??? P b .05 vs LSF.

⁺ P b .05 vs MSF.

Fig. 1. Observation of the pathological changes in ileal mucosa by light and electron mi- croscopy. A-E, Pathological sections from ileal mucosa of rats in different groups were stained with HE and examined by light microscopy (x100). A, Sham group, showing or- derly arranged ileal mucosal villi. B, CLP group, showing damaged ileal mucosal villi: the epithelial cells at the villus tips were wiped off, the subepithelial capillaries exhibited con- gestion, the central lacteals were expanded, the lamina propria was exposed and disintegrated, and blood capillaries were bleeding, with ulcer formation and widened in- tracellular tight junctions. C-E, Ileal mucosal structure in rats of the LSF, MSF, and HSF groups, respectively. The morphologies of ileal mucosal villi and intestinal glands were im- proved to varying degrees compared with panel B. The ileal mucosal morphology was more overtly improved in panel E than D, and in D than C. F-J, Pathological sections from ileal mucosa of rats in different groups were examined under by electron microscopy (x26,500). F, Sham group, showing orderly arranged ileal mucosal villi, with continuous tight junction between epithelial cells. G, CLP group, showing thinner and shorter ileal mu- cosal microvilli, with some disrupted, discontinuous tight junctions, and widened intercel- lular spaces. H-J, Ultrastructure of ileal mucosa epithelial cells of rats in the LSF, MSF, and HSF groups, respectively, showing less pronounced pathological changes: ileal mucosal microvilli were largely arranged in order and tight junctions were continuous, especially in the HSF group (J), where intercellular tight junctions were clearly visible.

Here, we showed that serum DAO and D-lactic acid levels are significant- ly increased in rats with sepsis, and SFI pronouncedly alleviated these symptoms, in a concentration-dependent manner. Taken together, SFI protects and maintains the intestinal mucosal epithelium.

Claudins are the most important components of tight junctions, with at least 24 members in mammals. Claudins form ion-selective channels in the paracellular pathway and are involved in the maintenance of tight

Fig. 2. Effect of SFI on Claudin-3 and ZO-1 expression levels in sepsis rats. A, Claudin-3 and ZO-1 levels in ileal tissues from Sham, CLP, HSF, MSF, and LSF groups, by Western blot. B, Quantification of panel A. Sham, sham operation group; CLP, severe sepsis group; LSF, low- dose SFI group; MSF, middle-dose SFI group; HSF, high-dose SFI group. n = 9-10. Data are mean +- SD. *P b .05 vs Sham; ^#P b .05 vs CLP; ^&P b .05 vs LSF; ^?P b .05 vs MSF.

junction permeability and cell polarization. Claudin-3, a 20-kDa protein, is found on both the apical side of tight junctions and the lateral plasma membrane. It is an indispensable component of tight junctions and plays important roles in maintaining epithelial phenotype [12]. Claudin-3 deficiency results in the translocation of intestinal bacteria and toxins.

It was recently shown that ZO-1 is involved in maintaining and regu- lating the epithelial barrier function and could also regulate the transport of cellular components. Zonula occludens protein-1 deficiency delays tight junction formation between epithelial cells and affects the produc- tion of attachment proteins [15], leading to the destruction of intestinal mucosal mechanical barrier and increased intestinal permeability.

In this study, HE staining and electron microscopy showed that the ileal mucosal villi were obviously damaged in rats with sepsis, with the epithelial cells at the villus tips wiped off. In addition, discontinuous intercellular tight junctions with widened gaps were obtained; the mi- crovilli were thinner and shorter, with some disrupted. After SFI

Fig. 3. Effect of SFI on mRNA expression levels of Claudin-3 and ZO-1 in sepsis rats. Sham, sham operation group; CLP, severe sepsis group; LSF, low-dose SFI group; MSF, middle- dose SFI group; HSF, high-dose SFI group. n = 9-10. Data are mean +- SD. *P b .05 vs Sham; ^#P b .05 vs CLP; ^&P b .05 vs LSF; ^?P b .05 vs MSF.

administration at different doses, the damage of ileal mucosal villi was improved: the villi were orderly arranged, with continuous tight junc- tions and narrower gaps. Furthermore, mRNA and protein expression levels of the tight junction proteins Claudin-3 and ZO-1 were signifi- cantly decreased in rats with sepsis; their levels were overtly higher after treatment with SFI, showing a dose-dependent effect. These find- ings indicate that SFI maintains the integrity of ileal mucosa’s epitheli- um and decreases the intestinal mucosal permeability by increasing Claudin-3 and ZO-1 expressions, thereby preventing the translocation of bacteria, endotoxin, and large molecular toxins into the blood stream and further preventing the aggravation of inflammation.

The mechanisms by which sepsis causes tight junction damage in in-

testinal mucosa’s epithelial cells are not well understood. However, it is believed to be closely related to oxidative stress caused by reactive ox- ygen species (ROS). Reactive oxygen species are continuously produced during the metabolic process of cells and tissues and include superox- ide, nitric oxide, hydrogen peroxide, and oxygen free radicals; they can peroxidate lipids by acting directly on cell organelles, which leads to DNA lesions and cell necrosis, further causing tight junction damage in epithelial cells [16,17]. Oxidative stress was shown to inhibit protein tyrosine phosphatase by activating tyrosine kinases, which leads to de- creased interaction between tight junctions and cell cytoskeleton [18]. Immunofluorescence analysis proved that both hydrogen peroxide and nitric oxide induce decreased tight junction protein expression and redistribution [19]. Hydrogen peroxide promotes calcium deple- tion, leading to adherens junction damage, accompanied with disrup- tion of tight junctions and increased permeability [20]. Hydrogen peroxide also induces the phosphorylation of tight junction proteins, such as occludin and ZO-1, thus destroying tight junctions [21]. The an- tioxidative effect under normal conditions prevents oxidative stress; however, excessive inflammation during severe sepsis may induce a pronounced oxidative stress and cause tight junction damage in the in- testinal mucosal epithelium. In addition, the oxygen demand of intesti- nal mucosal epithelial cells is particularly high, whereas the hypoxia caused by low tissue perfusion during sepsis results in abnormal intra- cellular metabolism, which affects tight junction Protein Synthesis. Our results indicated that the abnormal oxygen metabolism caused by severe sepsis leads to oxidative stress, which results in tight junction damage. After administration of SFI, the tight junction damage in ileal mucosal epithelial cells was improved in rats with sepsis. Therefore, we speculate that SFI might correct the abnormal oxygen metabolism of intestinal mucosal epithelial cells by improving tissue oxygen supply in severe sepsis, thereby restoring the damaged tight junctions in intes- tinal mucosal epithelial cells.

In this study, the functional changes in the liver and kidney as well as myocardial damage were also assessed in rats after SFI treatment, and no significant changes were observed, compared with the sepsis model group, indicating that SFI has no adverse effects on the heart, liver, and kidney. However, the toxicologic effects of SFI on other organs, including the spleen, lung, and thymus, were not assessed. This is a lim- itation of this study.

Overall, intraperitoneal administration of 5, 10, and 20 mL/kg SFI sig- nificantly alleviated the abnormal values for serum TNF-?, DAO, and D- lactic acid levels, and tight junction marker (ZO-1 and Claudin-3) gene and protein levels in ileal mucosal epithelial cells of rats with sepsis, re- storing the damaged tight junctions in ileal mucosal epithelial cells. These effects of SFI on sepsis parameters increased with its dose. How- ever, the detailed molecular mechanisms need further studies. It should be noted that a small sample size was used in this research, and the time point studied might have been too early to get the full picture.

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