Schisandrin A protects intestinal cells from mycophenolic acid-induced cytotoxicity and oxidative damage

Objectives: The gastrointestinal side effects of mycophenolic acid affect its ecacy in kidney transplant patients, which may be due to its toxicity to the intestinal epithelial mechanical barrier, including intestinal epithelial cell apoptosis and destruction of tight junctions. The toxicity mechanism of mycophenolic acid is related to oxidative stress-mediated the activation of mitogen-activated protein kinases (MAP K). Schisandrin A (Sch A), one of the main active components of the Schisandra chinensis, can protects intestinal epithelial cells from deoxynivalenol-induced cytotoxicity and oxidative damage by antioxidant effects. The aim of this study was to investigate the protective effect and potential mechanism of Sch A on mycophenolic acid-induced damage in intestinal epithelial cell. Methods: Caco-2 cells monolayers were treated with mycophenolic acid (10µM) and/or Sch A (10, 20 and 40µM) at 37°C for 24h, and cell viability was measured by MTT; Western blot and immunouorescence were used to detect the expression of relevant proteins. Intracellular ROS and apoptosis were measured by ow cytometry, and malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured by kits. Results: The results showed that Sch A signicantly reversed the mycophenolic acid-induced cell viability reduction, restored the expression of tight junction protein ZO-1, occludin and reduced cell apoptosis. In addition, Sch A inhibited mycophenolic acid-mediated MAPK activation and reactive oxygen species (ROS) increase. Conclusions: Sch A protected intestinal epithelial cells from mycophenolic acid intestinal toxicity, at least in part, by reducing oxidative stress and inhibiting MAPK signaling pathway. Conclusions: Sch A protected intestinal epithelial cells from mycophenolic acid intestinal toxicity, at least in part, by reducing oxidative stress and inhibiting MAPK signaling pathway.


Introduction
Mycophenolic acid (MPA) is an immunosuppressant commonly used as adjuvant therapy in kidney transplantation, which can effectively improve the long-term survival rate of the graft, but also has many adverse reactions [1]. Gastrointestinal adverse reactions such as vomiting and diarrhea are one of the most common adverse reactions [2]. The patients with gastrointestinal adverse reactions have to reduce the dose or even stop mycophenolic acid treatment, which lead to an increased risk of rejection and affect the outcome of the transplantation [3,4].
The intestinal toxicity of mycophenolic acid is related to the damage of the intestinal mechanical barrier, which is composed of intestinal epithelial cells and tight junctions between the cells [5]. Increased apoptosis of intestinal epithelial cells and altered expression of paracellular tight junction proteins (TJs) may both lead to mechanical barrier defects and cause intestinal symptoms such as diarrhea, gastrointestinal bleeding and enteritis [6,7]. TJs are important components of the intestinal epithelial barrier, which located at the most apical part of the epithelium, and consist mainly of transmembrane proteins (e.g., occludin, claudins) and peripheral proteins (e.g., ZO-1) [8]. Previous reports have shown that mycophenolic acid disrupts the intestinal mechanical barrier by altering the expression of TJs through activation ERK/p38/MLCK pathway [9,10]. Moreover, activated mitogen-activated protein kinases (MAPK) induces apoptosis by increasing Caspase 3 cleavage and Bax expression in intestinal cells [11].
It has been con rmed that activation of the MAPK signaling pathway in intestinal epithelial cells is related to the redox state of the cells. Oxidative stress-mediated increase in reactive oxygen species (ROS) can directly activate the MAPK [12]. MPA can cause dose-dependent mitochondrial dysfunction and increase the production of ROS [13]. Therefore, inhibition of MAPK activation regulated by oxidative stress may be a therapeutic target to alleviate the intestinal toxicity of MPA.
Our group's previous clinical observation found that diarrhea was signi cantly improved in patients who took mycophenolate mofetil (MMF) (a pre-MPA drug) in combination with WuZhi capsules. The main ingredient of WuZhi capsules is Schisandrin A (Sch A). Sch A is one of the main active components of Schisandra chinensis (turcz.) baill of the Magnoliaceae family, with various pharmacological effects such as antioxidant [14,15] Superoxide dismutase (SOD) activity analysis and malondialdehyde (MDA) content determination Caco-2 cells in logarithmic growth phase were inoculated into cell culture asks, fused and grown for one week, and treated with drugs for 24 h as described above. Washed three times with PBS, added lysate to collect cells, centrifuged at 1000r for 10 min to get the supernatant. BCA protein quanti cation kit (Beyotime Biotech, China) was used to detect protein content, and the nal treatment solution was added to 96-well plates according to the instructions of SOD and MDA assay kit (Jiancheng Bioengineering Institute, China), and the absorbance was detected at wavelengths of 450 nm or 530 nm. Then calculated SOD activity and MDA content according to the calculation formula.

Apoptosis Analysis
After the cell culture was completed, the cells were digested without EDTA, and the cells in the culture medium were collected. Centrifuge at 300g for 5 min in a centrifuge. Discarded the supernatant, added PBS to resuspend and centrifuge, repeated 2 times. Resuspended the cells with 400 μL of AnnexinV binding solution, added 4 μL of AnnexinV-FITC in the dark, and incubated for 15 min in the dark. Then added 7.5 μL of PI staining solution, mixed gently, incubated in the dark for 5 min, and then tested in the ow cytometer.

Statistical analysis
All data were statistically analyzed using Statistical Program for Social Sciences v.26.0 software (SPSS Inc., Chicago, IL, USA). The data was normally distributed and expressed by the mean standard deviation (SD). The difference analysis was carried out by one-way (ANOVA). All experiments were repeated at least three times. P < 0.05 was considered statistically signi cant. shown that MPA damages intestinal epithelial cells because it impairs the expression and distribution of TJs proteins [17]. Therefore, we tested the expression and distribution of two representative TJs proteins, ZO-1 and occludin in Caco-2 cell monolayer treated with Sch A and/or MPA. As shown in Fig. 2a, the results of Western blot showed that the expression of occludin and ZO-1 in Caco2 cells treated with MPA for 24h was signi cantly reduced compared with the control group, while after cotreatment with Sch A for 24h, the expression of ZO-1 and occludin increased in a dose-dependent manner. Similar results were obtained from immuno uorescence experiments. Sch A treatment can partially prevent the redistribution of ZO-1 and occludin induced by MPA and re-close the paracellular space. Overall, these data indicate that Sch A effectively protects the TJs of the intestinal epithelium damaged by MPA (Fig. 2b).

Results
Sch A reduces MPA-induced apoptosis in Caco-2 cells MPA has been reported to be related to the apoptosis of intestinal epithelial cells, so we detected the expression of apoptosis-related proteins. As shown in Fig. 3a, there was no signi cant change in the expression of the pro-apoptotic protein Bax, but compared with the control group, MPA reduced the expression of the anti-apoptotic protein Bcl-2 (P < 0.05). After treatment with Sch A for 24 h, MPA-induced apoptosis was signi cantly reduced by up-regulating Bcl-2 (P < 0.05 or P < 0.01). In addition, MPA increased the activation of Caspase-3, while Sch A inhibited the activation of Caspase-3 (P < 0.01). Subsequently, in order to further con rm whether the reduction of apoptosis is related to the protective effect of Sch A, we used Annexin V-FITC/PI staining to measure apoptosis. As shown in Fig. 3b and 3c, MPA signi cantly increased the early and late apoptosis rates of Caco-2 cells and reduced the percentage of normal cells (P < 0.05), and Sch A reversed the up-regulation of MPA on the apoptosis rate and increased the percentage of normal cells (P < 0.05).

Sch A inhibits MPA-induced activation of MAPK signaling pathway
To further explore the molecular signaling pathway of the protective effect of Sch A in the intestine, the effect of Sch A on ERK/JNK/p38 MAPK pathway in the MPA model was investigated by Western blot (Fig. 4).

Sch A reduces oxidative stress induced by MPA in Caco-2 cells
The imbalance between oxidation and antioxidant systems causes increased accumulation of ROS, oxidative stress, and oxidative damage to cells. In order to test whether Sch A reduces the oxidative stress induced by MPA in Caco-2 cells, rstly, we detected the production of ROS. The results of ow cytometry and H 2 DCFDA staining experiments showed that after MPA treatment, the ROS level in Caco-2 cells was signi cantly higher than that in control cells, while Sch A and MPA co-treatment signi cantly reduced ROS in cells (P < 0.05) (Fig. 5a-c). Next, we tested the activity of SOD and the content of MDA. As shown in Fig. 5d-e, compared with the MPA group, Sch A signi cantly increased SOD activity and reduced MDA content (P < 0.05).

Discussion
MPA is commonly used as an immunosuppressive agent in adjuvant renal transplantation, which effectively improves long-term graft survival, but the accompanying gastrointestinal adverse effects such as diarrhea greatly limit its use. It is now generally accepted that diarrhea caused by MPA may be due to damage to intestinal epithelial cells [18]. Our results suggest that Sch A can act as a protective agent against MPA-induced Caco-2 cell injury. The results of MTT assay showed that 10-50 µM of Sch A protected cells from MPA-induced cell damage and partially reversed the inhibition of cell viability by MPA. Therefore, this concentration range was selected for subsequent experiments.
The intestinal barrier includes three types of barriers: biological barrier, immune barrier and mechanical barrier. Mechanical barriers are important in maintaining intestinal function, which is a tight structure established by epithelial and endothelial cells interconnected through a tight junction (TJ) structure. Mechanical barriers effectively restrict the passage of bacteria and endotoxins [19,20]. Both increased apoptosis of intestinal epithelial cells and altered expression of paracellular tight junction proteins may lead to mechanical barrier defects in the intestinal epithelium, resulting in intestinal barrier dysfunction and causing diarrhea and enteritis [21]. "Tight junction" is an apical junctional complex (AJC) which consists of a variety of tight junction protein (TJs). It is reported that deletion of TJs induces increased paracellular permeability in vivo and in vitro, which in turn leads to diarrhea [22]. ZO-1 is an early identi ed cytoplasmic peripheral membrane protein of TJs, which consists of an amino-terminal and a carboxy-terminal half. Occludin is a transmembrane protein that binds to the amino-terminal of ZO-1 to form a tight junction complex. The carboxy-terminal of ZO-1 anchors this complex to the cytoskeleton by binding F-actin (cytoskeletal protein), which forms the paracellular barrier [23]. Our study showed that Sch A upregulated the expression of ZO-1 and occludin, and reduced the gap between intestinal epithelial cells.
In addition, apoptosis is one of the manifestations of MPA-induced intestinal epithelial cell damage [24,25]. A certain degree of apoptosis is necessary for intestinal epithelial cell proliferation and repair, but excessive apoptosis increases intestinal permeability and leads to intestinal barrier dysfunction, which in turn induces diarrhea [26,27]. Our results showed that apoptosis was increased after MPA treatment, and there was no signi cant change in Bax protein expression, but Bcl-2 protein expression was signi cantly downregulated, in addition to an increase in activated Caspase 3. In contrast, Sch A upregulated antiapoptotic protein expression, reduced Caspase 3 activation, and led to a signi cant decrease in the proportion of apoptotic cells. It indicates that Sch A inhibits MPA-induced apoptosis.
Although Sch A upregulates the expression of TJs and reduces apoptosis to protect intestinal epithelial cells, the mechanism is unclear. It has been reported that the activation of ERK/JNK/p38 MAPK signaling pathway downregulates TJs by increasing ELK1 phosphorylation and inducing the transcription of MLCK [28,29]; and increases apoptosis through the mitochondrial pathway [30,31]. And studies have con rmed that MPA regulates TJs through p38 MAPK [10]. Therefore, we then investigated the regulatory role of Sch A on the ERK/JNK/p38 MAPK signaling pathway. By in vitro Western blot experiments, we found that the regulation of the ERK/JNK/p38 MAPK signaling pathway by Sch A was evident, the expression of p-ERK, p-JNK and p-p38 were signi cantly down-regulated.
It has been con rmed that the increase of reactive oxygen species (ROS) mediated by oxidative stress in intestinal epithelial cells is the key factor which lead to intestinal epithelial cell apoptosis and downregulation of TJs expression [32,33]. Arsenite-induced downregulation of occludin in BEAS-2B cells via the ROS/ERK/ MLCK and ROS/p38 MAPK signaling pathways [9];ROS activates ERK1/2/MLCK pathway to result in endothelial cell tight junction de ciency and barrier dysfunction, leading to acute lung injury and death in CLP-induced septic mice [34] In summary, our study shows that Sch A plays a protective role against MPA-induced Caco-2 cell injury in vitro, and its protective effect on TJs is mediated, at least in part, by reducing oxidative stress and regulating the ERK/JNK/p38 MAPK signaling pathway. The signi cance of this experiment is the discovery of the protective effect of Sch A on MPA-induced intestinal cell injury, which provides a new research idea to improve the adverse effects caused by MPA related to intestinal permeability.

Conclusions
Sch A attenuated MPA-induced intestinal injury and reversed the inhibition of MPA on Caco-2 cell viability in an in vitro model. In addition, Sch A was found to inhibit MPA-induced oxidative stress, restore TJs expression and reduce apoptosis, possibly through the MAPK signaling pathway. These results suggest that Sch A may be a protective agent to ameliorate MPA-induced intestinal injury.  Figure 1 Effect of Sch A on MPA-induced cell viability of Caco-2 cells. Cells were treated with different concentrations of MPA and Sch A for 24h (a, b) and with 10 µM of MPA co-treated with different concentrations of Sch A for 24h (c). Cell viability was detected by MTT assay. The data were presented as mean ± SD, n = 3. *, **P < 0.05 and 0.01 signi cantly different from MPA treated cells, ##P < 0.01 signi cantly different from control. showed green uorescence, and DAPI-stained nuclei showed blue uorescence. The data were presented as mean ± SD, n = 3. *, **P < 0.05 and 0.01 signi cantly different from MPA treated cells, ##P < 0.01 signi cantly different from control. The bar represents 100 µm.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. FlowcytometryApoptosis.tif