The Inhibitory Impact of Schisandrin Against LPS-Induced Acute Kidney Injury in Vitro and in Vivo

Schisandrin (Sch) is a main bioactive component of Schisandra sphenanthera Rehd.et Wils. It has been reported that Sch could regulate in�ammatory disease. This study evaluated the anti-inammatory and anti-oxidant effects effect of Sch on lipopolysaccharide (LPS)-induced macrophages activation and acute kidney injury mice. Male Kunming mice were intraperitoneally injected with LPS (15 mg/kg) after administration of Sch (12.5, 25, 50 mg/kg) seven days for developing acute kidney injury vivo model. RAW264.7 macrophages were pretreatment Sch (10, 20, 40 µM) and administrated LPS (1 µg/ml) to create an in vitro injury model. ELISA results found that Sch administration reduced the production of in�ammatory factors induced by LPS in kidney tissues and RAW264.7 macrophages. It has been observed that Sch alleviated oxidative stress by reducing the levels of reactive oxygen species, myeloperoxidase and malondialdehyde, and increasing the activity of superoxide dismutase and glutathione peroxidase. Hematoxylin-eosin staining data suggested that Sch administration signi�cantly reduced in�ammatory cell in�ltration and the kidney tissue damage induced by LPS. The blood urea nitrogen and creatinine levels were also reduced by Sch treatment. In addition, Western blot and immunohistochemical analysis showed that Sch up-regulated the expression of Nrf2 and HO-1, and decreased the expression of p-p38, p-JNK, p-ERK1/2, p-IκBα, p-NF-κBp65 and TLR4. The current research showed that Sch reduced LPS-induced acute kidney injury by inhibiting in�ammation and oxidative stress, and provided insights into potential ways to treat AKI.


Introduction
Acute Kidney Injury (AKI) is a common clinical syndrome with kidney function declines rapidly, and accompanied with higher morbidity and mortality [1,2].AKI is mainly manifested as abnormal changes in serum creatinine (CRE), urea nitrogen (BUN) and glomerular ltration rate [3].In recent years, the incidence of acute kidney injury has been steadily increasing [4].It is more common in hospitalized and critically ill patients and seriously affects the prognosis of patients.It has also become a disease of public health issues affecting global society and economy [5,6].Therefore, it is very urgent to discover effective drugs for the treatment of acute kidney injury.
In ammation is a self-defensive physiological response of the immune system to injury and infection.
However, inappropriately activated in ammation can become pathogenic in ammatory diseases [7].In ammatory storm could be spotted in patients suffering from AKI with a large number of macrophages in ltrated in kidney tissues [8].Macrophages, as the crucial immune cells, secreted in ammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6), leading to the cascade of other in ammatory cytokines and production of excessive ROS, aggravating tissue damage [9,10].These indicated the crucial regulatory role of macrophages in the pathogenesis of AKI.Lipopolysaccharide (LPS), is a characteristic component of the cell wall of gram-negative bacteria, which was widely used to induce in ammation [11][12][13].When induced by LPS, the TLR4 dimer interacts with the downstream adaptor protein to induce MAPK and NF-κB signal transduction [13][14][15].Nuclear factor-κB (NF-κB) is a nuclear transcription factor necessary for the transcription and production of a large number of cytokines [16].After LPS stimulation, activated NF-κB signaling pathways promote the expression of interleukin-1β (IL-1β), nterleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and other proin ammatory cytokines [17].These factors promote the entry of macrophages and neutrophils into tissues and organs, causing in ammation [17].In addition, as one of the most typical signal transduction cascades, the MAPK pathway such as JNK, ERK and p38 MAPK [18] is also involved in the production of various in ammatory mediators in the process of in ammation [19].Moreover, LPS stimulation induces excessive oxidative stress [20].The multifunctional regulator nuclear factor erythroid 2 related factor (Nrf2) is a main regulator of cell redox homeostasis activating the expression of antioxidant enzyme genes, such as blood oxygenase-1 (HO-1) [21].Mounting studies reported that the up-regulation of HO-1 and its products can reduce the production of ROS and in ammatory cytokines in lipopolysaccharide (LPS) stimulated macrophages, thereby regulating oxidative damage and in ammation [22][23][24].
Schisandra is the dry and mature fruit of Schisandra sphenanthera Rehd.etWils. of the magnolia family Schisandra sphenanthera [25].As the main active component of schisandra (see Fig. 1 for the structure), schisandrin (Sch) has multiple effects such as antioxidant, anti-in ammatory and detoxi cation [26].
However, the effect and mechanisms of Sch on macrophage activation and acute kidney injury remain unclear.Therefore, the study utilized LPS-induced macrophage and acute kidney injury (AKI) mice.The results demonstrated that Sch pretreatment ameliorated LPS-induced macrophage activation and AKI through inhibiting in ammation and oxidative stress.

In Vitro
2.2.1 Cell culture RAW 264.7 macrophages were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS) in an incubator at 37°C and 5% CO2.In the AKI group, the cells were treated with LPS (10 µg/mL) for 24 h.Sch was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and adjusted to the nal concentrations (10, 20 and 40 µM) using complete DMEM medium.Before LPS treatment, the administration group received different doses of Sch treatment.

Cell viability assay
MTT was used to detect the effect of different concentrations of Sch on RAW 264.7 macrophages to select the appropriate concentration.Cells in good condition were inoculated into a 96-well plate (1×104/well), and incubated with 180 µL of medium.After 24 h, the cells were treated with Sch (0, 1.25, 2.5, 5, 10, 20, 40, 80, and 160 µM) and cultured for 24 h.Carefully removed the supernatant from the well plate, and added 20 µL MTT solution (0.5 mg/mL) to each well and incubated for 4 h.The purple MTT formazan was solubilized with 180 µL DMSO/well, and measured the OD value at 490 nm by a microplate reader (BioTek ELx800, USA).

IL-6 and monocyte chemotactic protein-1 (MCP-1) production in RAW 264.7 macrophages
The release of IL-6 and MCP-1 was determined using the ELISA assay kit.In brief, the RAW246.7 cells were cultured in 96-well plates and incubated with in DMEM containing different concentrations of Sch and LPS for 12 h respectively.Using a microplate reader to determine the OD value of cytokine levels according to the manufacturer's instructions.

2.2.4
Measurement of ROS levels in RAW 264.7 macrophages RAW264.7 macrophages were treated with different concentrations of Sch (10, 20, 40 µM) for 24 h, and then incubated with LPS (1 µg/ml) for 24 h.After discarding the cell culture supernatant, 10 µM DCFH-DA was added and incubated at 37°C for 1 h.After washing several times with PBS, the intracellular reactive oxygen species production was observed and imaged using a uorescence microscope (Nikon, Tokyo, Japan).

In Vivo
Male Kunming mice (18-22 g, n = 60) with an average age of 8 weeks were purchased from the Animal Experiment Center of Xi'an Jiaotong University (Shanxi Province, China).Before treatment, all mice had free access to water and food in the animal room, and adapt to the environment under pathogen-free conditions of 12 h of light and 12 h of darkness every day.There were ve animals in each cage.This study was conducted in accordance with the experimental protocol approved by the Institutional Animal Genetics Committee of Xi'an Jiaotong University.

Mice model of LPS-induced AKI and drug administration
AKI was induced by LPS.After a three-day adaptation period, the mice were randomly divided into six experimental groups (ten mice in each group), namely control group, AKI group (LPS with 15 mg/kg), Sch (12.5, 25 and 50 mg/kg) + LPS group, and DEX + LPS group.Sch was dissolved in 0.1% Tween and prepared dosing solutions with nal concentrations of 12.5 mg/kg, 25 mg/kg and 50 mg/kg using 2% sodium carboxymethyl cellulose.Before the administration of LPS, mice in the (Sch + LPS) group and (DEX + LPS) group were fed Sch and DEX for 7 days, respectively.On the seventh day, 6 h after intraperitoneal injection of LPS (except the control group), blood was taken from the eyeballs to determine factors, and then the mice were sacri ced and two kidneys were taken out for further analysis (hematoxylin-eosin (H&E) staining and immunohistochemistry test).

Determination of in ammatory cytokines in the tissue
The levels of TNF-α, IL-6, IL-1β in the rice serum were detected by ELISA kits.According to the manufacturer's agreement, continuously added working reagents, and readed the optical density (OD) of each microcell at 450 nm with a microplate reader (Bio-Tek ELx800, USA).

Assay of tissue CRE, BUN and Serum Cystatin C (sCysC)
Kidney function was assessed by CRE, BUN and sCysC using commercial kits purchased from Nanjing Jiancheng Institute of Biological Engineering.According to the instructions of the kit, 100 mg of tissue was weighed and physiological saline was added to prepare a 10% tissue homogenate.Used the corresponding working solution in turn and incubated at 37°C, and then measured the optical density (OD) at 450 nm in a microplate reader (Bio-Tek ELx800, USA).

Oxidative stress in the kidney
To evaluate the oxidative stress and antioxidant markers in kidney tissue of different treatment groups.According to a certain ratio, the kidney tissues were dissolved in physiological saline to prepare a 10% tissue homogenate.The levels of MDA, MPO, SOD and GSH-Px were measured by the instructions of the test kit to assess the tissue damage of the kidney.

Histopathological examination
For routine analysis, the collected fresh kidney tissues were xed with 4% paraformaldehyde for 48 h, and dehydrated with different concentrations of ethanol and xylene.Next, the sample embedded in para n at 60°C, and sliced into 5 um thick sections.Then, the tissues were depara nized and stained with H&E according to standard histopathology protocol.The degree of kidney damage was observed under an optical microscope (Olympus Optical, Tokyo, Japan).Moreover, 0 means no damage; 1 means mild damage; 2 means moderate damage; 3 means severe damage; 4 means that the degree of damage is very severe as the standard to evaluate kidney damage.

Immunohistochemistry
Using immunohistochemistry experiments to evaluate protein expression in kidney tissue.The kidney tissue sections were depara nized and rehydrated with xylene, absolute ethanol, double distilled water and PBS, and antigen retrieval solution (citrate buffer) was added for antigen retrieval.Then, 3% H2O2 was used to block the activity of endogenous peroxidase and incubated with goat serum for 10 minutes at 37°C for blocking.The tissue sections were incubated with phosphorylated JNK1/2, HO-1, and NF-κBp65 antibodies overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at 37°C.Staining with DAB and counterstain with hematoxylin.The sections were observed using an Olympus DP-71 microscope equipped with a digital camera.Finally, the results were semi-quantitatively evaluated based on the percentage of positive cells in ten random images viewed at x200 magni cation.

Western blotting
The quanti ed kidney tissue was lysed with RIPA lysis buffer to prepare a tissue homogenate and the supernatant was collected by centrifugation (1000 r/min).The BCA kit were used to detect the protein concentration to determine the loading amount.Separated the same amount of protein with a 10% SDSpolyacrylamide electrophoresis gel, and then transferred it to a PVDF membrane.After blocking with 5% fat-free milk powder at room temperature for 2 h, incubated the membrane with primary antibodies overnight (4°C).The next day, the blots were incubated with the secondary antibodies for 1 h (37°C) and used the ECL blot analysis kit to generate a positive signal.Analyze and quantify the intensity of protein bands by luminescence imaging system to detect total protein density.

Statistical analyses
Statistical analysis was performed using Graph Pad Prism 5.0 and all data were expressed as mean ± SEM.A one-way analysis of variance (ANOVA) and Dennett post-inspection were used to determine the statistical difference between multiple groups with P < 0.05 as the standard.

Effect of Sch on the toxicity of RAW 264.7 macrophages
The MTT assay was used to detect the effect of Sch on the survival rate of RAW264.7 macrophages.The results (Fig. 1B) showed that compared with the control group, when the cells were pretreated with Sch of 1.25 to 80 µM, there was no signi cant change in cell viability.However, Sch at 160 µM signi cantly inhibited cell viability (P < 0.01).Therefore, we decided to choose 10, 20, and 40 µM Sch for subsequent cell research.

Effect of Sch on LPS-induced RAW264.7 macrophages cytokine production
As shown in Fig. 1C and 1D, the effect of Sch on the expression of in ammatory factors IL-6 and MCP-1 in RAW264.7 macrophages.To verify the effect of Sch in vitro, LPS were used to process RAW264.7 macrophages.Compared with the control group, the levels of IL-6 and MCP-1 in cells were signi cantly increased after LPS stimulation (P < 0.01).However, compared with the high expression (160 ± 1.79 pg/ml, 450 ± 21.6 pg/ml) in the LPS group, the expression levels of IL-6 and MCP-1 decreased to 87.0 ± 9.58 pg/ml and 216 ± 5.81 pg/ml at the Sch dose of 40 µM, respectively (P < 0.001).The results showed that Sch has an inhibitory effect on the production of IL-6 and MCP-1 in RAW264.7 macrophages stimulated by LPS.
3.3 Effect of Sch on the activation of ROS in RAW264.7 macrophages ROS are widely known to be related to the intensity of oxidative stress in cellular responses.In the AKI group, the green uorescence intensity increased signi cantly, indicating that the production of ROS in the cells increased (P < 0.001).The production of ROS in the administration group was signi cantly reduced (P < 0.01), which indicated that Sch was able to inhibit the production of oxidative stress by suppressed the activation and production of ROS (Fig. 2A-B).

Effect of Sch on Nrf2/HO-1 signaling pathway in RAW264.7 macrophages
The Nrf2 pathway is involved in the occurrence and development of cellular oxidative stress.It was still unknown if Nrf2 pathway was involved in the roles of Sch in LPS-stimulated RAW264.7 macrophages.Therefore, western blot was performed to further explore the effect of Sch and Nrf2 inhibitor luteolin on Nrf2 /HO-1signaling pathway-related proteins expressions.From the results, we found that Sch treatment signi cantly increased Nrf2 and HO-1 expressions, while luteolin inhibited the production of these proteins.In addition, Sch still up-regulated the expression of Nrf2 and HO-1 under the premise that luteolin exerts an inhibitory effect.These data means that Sch can inhibit the production of LPS-induced oxidative stress in RAW246.7 cells by participating in the activation of Nrf2/HO-1.

Effects of Sch on the content of CRE, BUN and sCysC in the kidney tissues
To roughly assess the kidney damage in mice, the content of BUN and CRE in the kidney tissue were measured.As shown in Fig. 4A, in the LPS-induced AKI group, the levels of CRE were signi cantly increased by 3-fold (P < 0.001) compared with the control group.On the other hand, compared with the LPS group, the administration group (receiving 50 mg/kg of Sch pretreatment) reduced the activity of CRE in mice kidney tissue by 35.2 µmol/L (P < 0.001).Compared with control mice (27.9 ± 0.158 mmol/L), LPS signi cantly enhanced the activity of BUN (54.3 ± 2.60 mmol/L) in mice (P < 0.001), while BUN activity was signi cantly down-regulated at the Sch concentration of 12.5, 25 and 50 mg/kg (P < 0.01, Fig. 4B).
At the same time, changes in sCysC levels were also tested in experiments to assess the effect of Sch on kidney function.As shown in Fig. 4C, LPS induced overexpression of sCysC (P < 0.01), which increased 0.477 ng/ml compared to the control group.However, Sch and DEX pretreatment signi cantly reduced the level of sCysC in mice serum (P < 0.05).Moreover, this study also used H&E staining to assess the degree of kidney damage.As shown in Fig. 4D, in the kidneys of the LPS group, in ammatory cell in ltration, disappearance of brush border and destruction of glomerular structure were observed.However, the pretreatment with Sch at doses of 12.5, 25, or 50 mg/kg and DEX at 5 mg/kg signi cantly reduced the pathological changes caused by LPS.In general, Sch pretreatment can effectively inhibit the production of these in ammatory factors by LPS.

Effect of Sch on the MDA, SOD, MPO and GSH-Px levels in the kidney tissues
Kunming mice were pretreated with different concentrations of Sch and a certain dose of DEX, and then stimulated with 25mg/kg LPS for 6 h.As shown in Fig. 5A-B, GSH-Px and SOD levels activity were decreased in the LPS-induced AKI group (P < 0.05).In contrast, pretreatment with Sch (12.5, 25, and 50 mg/kg) and DEX signi cantly increased the SOD and GSH-Px content (P < 0.05).In addition, the activity of MDA and MPO were detected in kidney tissue.Compared with the control group (1.06 ± 0.048 nmol/mg prot and 0.234 ± 0.0078 U/g), mice tissues in the LPS group (P < 0.01) had higher levels of MDA (2.68 ± 0.023 nmol/mg prot) and MPO (0.314 ± 0.0029 U/g).However, the Sch group showed a signi cant downregulation of the activities of MDA and MPO by 0.09734 U/g, 0.1298 U/g, 0.1534 U/g (P < 0.001) at concentrations of 12.5, 25 and 50 mg/kg (Fig. 5C-D).

Sch activated the Nrf2/HO-1 signaling pathway in the kidney
This experiment evaluated the role of Nrf2/HO-1 pathway in kidney protection.In order to verify the expression of Nrf2 and HO-1 in mice kidney tissue induced by LPS, it was pretreated with different concentrations of Sch and DEX, then stimulated with LPS and measured protein expression (Fig. 6A).
Induction of LPS resulted in a signi cant decrease in the expression of Nrf2 and HO-1 protein compared to the control group (P < 0.01).On the contrary, the protein levels of Nrf2 and HO-1 in the administration of Sch (50 mg/kg) showed an apparent increase, and the expression of HO-1 approached the level of the control group (P < 0.05).In addition, the results of immunohistochemistry further showed that pretreatment with Sch can boost the expression of HO-1 protein (P < 0.05, Fig. 6B).Our research showed that Sch can inhibit the activation of Nrf2/HO-1 signaling pathway induced by LPS.

Sch inhibited NF-κB and MAPK proteins expression in the kidney
NF-κB signaling pathway plays a key role in the in ammatory response.This study tested the expression of NF-κB signaling pathway.The results of the Western blot in Fig. 7A showed that the expression of p-NF-κBp65, p-IκBα and TLR4 in the LPS group were signi cantly higher than that in the control group, increasing by 50%, 63% and 68% (P < 0.001).By contrast, the protein concentration after administration of Sch was decreased (P < 0.01).The results showed that Sch reduced the expression of NF-κB pathway protein stimulated by LPS.In order to further investigate the anti-in ammatory effects of Sch, western blot was also performed to detect the expression of key proteins related to the MAPK pathway.LPS stimulation signi cantly upregulated the levels of p-JNK1/2, p-ERK1/2 and p-P38 (P < 0.001), and Sch treatment attenuated this change.At the dose of Sch was 50 mg/kg, the protein expression of p-ERK and p-JNK decreased to a level close to that of the control group (P < 0.05, Fig. 7B).Immunohistochemical analysis further proved the role of Sch.These results showed that Sch effectively reduced the proteins expression (P < 0.01) of the NF-κB and MAPK pathway in kidney tissue (Fig. 8).

Discussion
In ammatory diseases are known to be caused by excess in ammatory responses and oxidative stress [27].In this study, we described the anti-in ammatory and anti-oxidative activity of Sch in LPS-induced macrophage and acute kidney injury in mice.
LPS administration may induce systemic in ammation reaction, and increase the number of neutrophils and accelerate the release of cytokines and and in ammatory mediators ,including IL-1β, IL-6, MCP-1 and TNF-α [28].Their overproduction can amplify in ammation and cause tissue damage [29,30].Moreover, CRE, BUN and sCysC are widely used as markers to assess kidney function, which were used for early detection of AKI [31,32].CRE, BUN and sCysC levels tend to increase due to kidney abnormalities induced by LPS administration [33].In addition, LPS also induce kidney pathological alterations, such as in ammatory cell in ltration.In this study, we found that the levels of these pro-in ammatory mediators in LPS-induced RAW264.7 cells and acute kidney injury mice were signi cantly decreased in Sch pretreatment group.Additionally, Sch signi cantly reduce the increased levels of BUN, sCysC and CRE in the mice with LPS stimulation.We also found that Sch clearly relieved in ammatory in ltration of kidney tissues.Based on the above results, it may be concluded that Sch plays an anti-in ammatory effect by inhibiting the expression of pro-in ammatory factors and kidney tissue damage, and then controls the activation of macrophages and the progression of acute kidney injury.
According to literatures, NF-κB and MAPK are involved in the occurrence and development of innate immunity and in ammation, and also regulated the production of numerous pro-in ammatory chemokines and cytokines [34].To study the mechanism of Sch in regulating LPS-induced kidney injury, we detected components of MAPK and NF-κB signaling pathways.In the present study, NF-κB pathway were notably activated by LPS in kidney tissues.However, Sch pretreatment decreased the expression levels ofp-NF-κBp65 and p-IκBα and TLR4.This indicated that NF-κB signaling activation mediates the anti-in ammatory effect of Sch in AKI mice.Moreover, the activation of MAPKs also promote the release of large number of in ammatory mediators [35].Among them, the ERK signaling pathway is involved in cell proliferation and differentiation, while the JNK and p38 MAPK pathways mainly mediate proin ammatory mediators and regulate cell apoptosis [36].Therefore, this study examined whether Sch can block the MAPK in ammatory pathway in mice with LPS-induced acute kidney injury.As expected, activated p-p38, p-ERK1/2 and p-JNK1/2 were partially down-regulated by Sch.In general, pretreatment of Sch attenuated TLR4-mediated activation of MAPKs and NF-κB, and further modulated the production of pro-in ammatory mediators and reduced kidney damage to inhibit the in ammatory response of AKI.
Oxidative stress are the key pathophysiological processes in the progress of kidney injury [37].Studies have reported that LPS could promote the up-regulation of oxidative stress in tissues [38].The formation of MDA and the enhancement of MPO activity will accelerate the in ltration of neutrophils in the kidney tissue and promote the progress of in ammation [38] [39].As effective antioxidants, SOD and GSH-Px are equally important in response to oxidative stress and regulating the redox state of cells [37,[40][41][42].Studies also showed that Nrf2, an important antioxidant pathway, protect cells from in ammation through regulation of oxidative stress [43].It also regulates the expression of the antioxidant enzyme HO-1 in macrophages [44].The up-regulation of HO-1 can reduce the release of ROS and in ammatory cytokines, thereby protecting tissues from damage [23].Our data showed that Nrf2 protein expression increased after Sch pretreatment and activated the Nrf2/HO-1 pathway, which upregulated the expression of downstream antioxidant HO-1 protein.Moreover, Sch also prevented the LPS-induced decreases in SOD and GSH-Px levels and the increases in MDA and MPO levels in the kidney tissue of AKI mice.These results indicated that Sch can reduce the kidney damage caused by oxidative stress through upregulating the Nrf2/HO-1 signaling pathway.In the current study, we also observed that Sch inhibited production of ROS in LPS-treated RAW264.7 cells at a dosage-dependent manner.These data further provided the evidence for the protective effect of Sch in LPS-induced AKI, namely the anti-oxidative stress effect of Sch.
In conclusion, Sch have a great preventive effect on LPS-induced AKI.These bene cial effects are closely related to anti-in ammatory and anti-oxidative stress pathways.These ndings strongly suggest that Sch could be a potential medicine for the treatment of LPS-induced AKI in the future.However, there are some limitations in this paper.The LPS-induced AKI mice model could not mimic all aspects of the human conditions and need further studies to the potential role of Sch in LPS-induced human kidney macrophages.

Declarations Data Availability
The      The expression of oxidative stress pathway related proteins in LPS-induced acute kidney injury mice.(A) Western blotting showed the expression levels of Nrf2, HO-1 and β-actin in the kidneys of mice.The treatment of Sch (12.5, 25, 50 mg/kg) and DEX (5 mg/kg) was continued for 7 consecutive days, and LPS was administered 1 h after the administration on the last day.All mice were treated 4 h later to receive tissues.The kidney was prepared as a tissue homogenate for Western blot analysis.Among them, β-actin was used as internal reference proteins.(B) Sch alleviated LPS-induced oxidative stress by immunohistochemical analysis.Magni cation ×200.The data is expressed as the relative ratio of the speci ed protein to the internal reference protein.This part of the experiment was repeated three times.Signi cant difference on statistics are *P < 0.05; ***P < 0.001 vs. the control mice; ###P < 0.001 vs. the LPS-induced mice.The effect of Sch on the expression of p-NF-κBp65 and p-ERK1/2 proteins in kidney tissue was investigated by immunohistochemistry.After 1 h of intragastric administration, LPS was stimulated for 4 h, and all mice were sacri ced to obtain kidney tissue.The concentrations of (A) p-NF-κBp65 and (B) p-ERK1/2 were collected.Magni cation ×200.The brown part is a sign of a positive reaction.Data are presented as the mean ± SEM (n=3).Compared with the control group, the signi cant differences were ###P < 0.001, compared with the LPS-induced model group, the signi cant differences were *P < 0.05, **P < 0.01 and ***P < 0.001, respectively.
chemical structure of schisandrin.

Figure 2 Effect
Figure 2

Figure 3 Effect
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Figure 4 Effect
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Figure 5 The
Figure 5