Matrine combined with Osthole inhibited the PERK apoptosis of splenic lymphocytes in PCV2-infected mice model

doi:10.21203/rs.3.rs-627445/v1

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Matrine combined with Osthole inhibited the PERK apoptosis of splenic lymphocytes in PCV2-infected mice model

https://doi.org/10.21203/rs.3.rs-627445/v1

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Background

PCV2 (Porcine circovirus type 2) is one of the major pathogens commonly in pigs, which can cause immunosuppression and apoptosis. Vaccinations and single drugs are not totally prevent and treat PCV2 diseases. We have previously reported that the synergistic anti-PCV2 effects of Matrine and Osthole were better than Matrine or Osthole alone in vitro, Matrine and Osthole were purchased with a clear content, chemical structure and plant origin. This study aimes to evaluate theirs synergistic anti-PCV2 effect and mechanism in Kunming (KM) mice model infected with PCV2. KM mice were randomly divided into 8 groups, namly: normal control group, PCV2 infected group, Matrine combined with Osthole high dose treatment group (40 mg/kg + 12 mg/kg), medium dose treatment group (20 mg/kg + 6 mg/kg), low dose treatment group (10 mg/kg + 3 mg/kg), Matrine treatment group (40 mg/kg), Osthole treatment group (12 mg/kg) and Ribavirin positive control group (40 mg/kg). PCV2 was intraperitoneally (i.p.) injected in all mice except the normal control group. At 5 days post-infection (dpi), mice in different treatment groups were injected i.p. with various doses of Matrine, Osthole and Ribavirin once daily for 5 consecutive days.

Results

The synergistic inhibition effect of Matrine and Osthole on PCV2 replication in mouse liver was significantly stronger than that of Matrine and Osthole alone. The protein expression of GRP78, p-PERK, p-eIF2α, ATF4, CHOP, cleaved caspase-3 and Bax were significantly reduced, but the protein expression of Bcl-2 was significantly increased in Matrine combined with Osthole groups, which alleviated the pathological change caused by PCV2, such as interstitial pneumonia, loss of spleen lymphocytes, infiltration of macrophages and eosinophils.

Conclusions

The synergistic effect of anti-apoptosis was better than that of Matrine and Osthole alone, although both of Matrine and Osthole could also directly inhibited the expression of PCV2 Cap and then inhibited the apoptosis of spleen cells induced by PCV2 Cap through the PERK pathway activated by endoplasmic reticulum (ER) GRP78. These results provide a new insight into controlling PCV2 infection and provide good component prescription candidate for the development of novel anti-PCV2 drugs.

Virology

Matrine

Osthole

PCV2

GRP78

apoptosis

PERK

PCV2 is an important pathogen of Post-weaning Multisystemic Wasting Syndrome (PMWS) and induces immunosuppression and apoptosis in the infetced piglet [1], which has become one of the most economically important diseases to the global pig industry [2]. The PCV2 vaccine effectively reduced the virus infection in pigs since it was widely used in 2007. However, the PCV2 strain is prone to mutation and cannot be best matched with existing vaccines, resulting in the inability of vaccine immunity to eradicate PCV2 infection [3, 4]. Sometimes the mutation of virus genes can be accelerated [5]. Therefore, it is urgent need to develop anti-PCV2 drugs to prevent and mitigate injury. In recent years, researches are increasingly concentrated on the anti-viral activities of natural compounds due to their characteristics of safety, high efficiency, low toxicity and low residue, as well as their unique clinical efficacy. PCV2 is a DNA virus that causes immunosuppression. Many viruses have developed complex mechanisms to interact with the host immune system to evade immunity after co-evolving with the host, these mechanisms provide cellular targets for antiviral intervention [6]. ER is an important site for viral replication and matur-ation. The up-regulation of the molecular chaperone GRP78 is an ER stress marker [7]. According to Zhou et al [8]. in PK-15 cells infected with PCV2, Cap caused endoplasmic reticulum stress (ERS), induced the expression of the stress marker protein GRP78, initiated the unfolded protein response to the selective activation of the PERK/eIF2a/ATF4/CHOP apoptosis pathway, and promoted the apoptosis of PK-15 cells.

Matrine is an alkaloids and the main bioactive component of sophora. It has anti-inflammatory [9], anti-cancer [10], anti-virus [11, 12], anti-apoptosis [13, 14], and other biological activities. Osthole is the main bioactive ingredient of Fructus Cnidii and has anti-inflammatory [15], anti-cancer [16], anti-virus [17], anti-immunosuppression [18] and anti-apoptosis effects[19]. Our previous studies have showed that Matrine combined with Osthole has a synergistic anti-PCV2 effect and anti-apoptosis induced by PCV2 Cap in vitro [13]. However, due to the complexity of clinical treatment mechanism and animal body function, the anti-PCV2 effect of Matrine combined with Osthole in vivo and its mechanism are still unclear.

Therefore, we choose Matrine and Osthole as the active ingredients of Traditional Chinese medicine according to the guidance of traditional Chinese veterinary theories, a component prescription with clear chemical structure, purity, specific biological and various pharmacological activities is formed. And to explore the synergistic anti-PCV2 effect and mechanism of Matrine combined with Osthole in a PCV2 infected mouse model.

Establishment of KM mice model infected with PCV2

PCR results showed that the PCV2 Cap gene were detected from the liver, thymus, spleen, inguinal lymph nodes, lung and blood of mice at 5 dpi with PCV2, but not in the mormal control mice (Fig. 2a, the original images see Additional file 1). The results indicated that the KM mouse model of PCV2 infection had been successfully established and the whole body of the mice was in the state of viral infection.

The results of HE staining showed that, compared with the normal control mice, the lungs of mice infected with PCV2 for 5 days showed the typical features of interstitial pneumonia, such as significantly widened interstitium and significantly reduced alveolar space (Fig. 2b, the original images see Additional file 1). Therefore, The lung was used to evaluate the effect of Matrine combined with Osthole on the pathological injury of mice infected with PCV2 in subsequent experiments. The results of qPCR showed that the expression of Cap gene was detected in the liver, thymus, lungs, spleen and lymph nodes of mice infected with PCV2, and the expression of Cap gene was the highest in the liver (P<0.05) (Fig. 2c). Therefore, liver was selected as the target organ and the expression levels of PCV2 Cap gene and protein in liver were analysed for the assessment of the synergistic anti-PCV2 effect of Matrine and Osthole.

It was revealed that there was no significant difference in body weight gain and lung index between normal control and PCV2 challenged mice (P>0.05). However, the spleen index in PCV2 challenged mice was significantly increased, compared with the normal control group (P<0.05) (Figs. 2d-f). Therefore, the mice spleen was used as the target organ to investigate the synergistic effect of Matrine and Osthole on the pathological change caused by PCV2 infection and the mechanism of anti-PCV2.

PCV2 replication in mouse liver

In order to explore the PCV2 replication in mouse, qPCR was used to detect the expression of Cap genes in mice liver infected with PCV2 at d 8, 11 and 14 was detetmined by qPCR. The results showed that the viral load in the liver of the PCV2 infected group was the highest when the mice were infected with PCV2 for 11 d (d 6 after compounds adminstration) (Fig. 3). Therefore, this conclusion provides a base for selecting the time points to study the synergistic anti-PCV2 effects of Matrine combined with Osthole.

Inhibition of the PCV2 replication in mouse liver by Matrine combined with Osthole

The expression of Cap gene in the liver of PCV2 infected mice at d 8, 11 and 14 (d 3, 6 and 9 after compounds adminstration) was detected by qPCR.

The results in Figs. 4a-c showed that the expression of Cap genes was significantly increased in the PCV2 infection group compared with the normal control group (p<0.05). Compared with the PCV2 without treatment group, the expression of Cap gene was significantly reduced in the high dose (40 mg/kg+12 mg/kg) and medium dose (20 mg/kg+6 mg/kg) of combined groups (Fis. 4a-c). There was no significant difference in the expression of Cap genes in the combined high, medium and low dose groups compared with the Matrine, Osthole and Ribavirin alone groups (Fig. 4a, p<0.05). But the expression of Cap gene was significantly reduced in the high and medium dose combined groups compared with the Matrine and Osthole alone groups (p<0.05), there was no significant difference in the expression of Cap genes in the low dose combined group (Fig. 4b, P>0.05). Compared with the Matrine, Osthole and Ribavirin groups alone, the expression of Cap gene was significantly reduced in all combined groups except for the low dose one (Fig. 4c, p<0.05).

In conclusion, the replication of PCV2 Cap in the mice liver were inhibited by the combined treatment of Matrine and Osthole. In addition, the results in Fig. 3 shown that “the viral load of PCV2 infected mouse liver was the highest at 11 days after infection”. Therefore, mice infected with PCV2 for 11 d (d 6 after compounds adminstration) were selected for subsequent studies.

Figs. 4d and 4e (the original blot images involved see Additional file 2.) showed that, compared with the PCV2 infection without treatment group, the expression of Cap proteins in all treatment groups with compounds were significantly reduced (P<0.05). The expression of Cap proteins was significantly lower in the high dose combined group than Matrine, Osthole and Ribavirin alone groups (p<0.05). And compared with theMatrine group of 40 mg/kg, the expression of Cap protein was significantly reduced in the medium and low dose combined groups (P<0.05), but there was no significant change when compared with Osthole group and Ribavirin alone groups (P>0.05).

In short, the synergistic anti-PCV2 effect of Matrine combined with Osthole at 40 mg/kg+12 mg/kg was demonstrated.

The effect of Matrine combined with Osthole on lung and spleen infected by PCV2 in mice

Representative images of HE staining results of lung and spleen tissues of mice infected with PCV2 at d 11 after PCV2 infection were shown in Fig. 5 compared with the lungs of mice in the normal group, the alveolar septum in the PCV2 group was widened and the alveolar cavity was narrowed significantly indicating the typical symptoms of interstitial pneumonia. The pulmonary pathological changes induced by PCV2 was alleviated in all compound treated groups, among which Matrine and Osthole combined treatment showed better effect (Fig. 5a , the original HE staining images see Additional file 3). The deletion of lymphocytes and infiltration of macrophages and eosinophil was observed in the spleen of PCV2 infected mice compared with the normal control mice. The pathological change of spleen induced by PCV2 was improved significantly in all compound treatments groups, especially in the Matrine and Osthole combined treatment group (Fig. 5b, the original HE staining images see Additional file 4).

Matrine combined with Osthole inhibited the apoptosis of spleen lymphocytes in PCV2 infected mice

The results of IHC showed that the expression of cleaved caspase-3 protein in the spleen was increased in the PCV2 group at d 11 (d 6 after compounds adminstration) of PCV2 infection compared with that in the normal control group. Compared with the PCV2 without treatment group, the expression of cleaved caspase-3 apoptin in all treatment groups was decreased (Fig. 6a, the original IHC images involved see Additional file 5).

The results of Western blot showed that, compared with normal controls, the expressions of cleaved caspase-3 and Bax were significantly increased (p<0.05) while the expression of Bcl-2 was significantly decreased in PCV2 infected mice at d 11 (p<0.05). Compared with the PCV2 group, the expression of cleaved caspase-3 and Bax in all the treated groups were significantly reduced (p<0.05), but the expression of Bcl-2 were significantly increased (p<0.05). The expression of cleaved caspase-3 was significantly reduced and the expression of Bcl-2 was significantly increased in the high dose combined group compared with the Matrine, Osthole and Ribavirin alone groups (p<0.05). The expression of Bax in the high dose combination group was significantly reduced, compared with the groups of Matrine and Ribavirin alone groups (p<0.05), but no significant difference was found between the Osthole alone group and the high dose combination group (p>0.05) (Figs. 6b-e, the original blot images involved see Additional file 6).

Apoptosis of spleen lymphocytes induced by PCV2 was inhibited by Matrine combined with Osthole through the pathway of PERK mediated by GRP78

The results of Western blot (Fig. 7, the original blot images involved see Additional file 6) showed that the expressions of pro-apoptins GPR78, p-PERK, p-eIF2α, ATF4 and CHOP were significantly increased in the PCV2 infected mice at d 11 compared with the normal control group (p<0.05). Compared with the PCV2 without treatment group, the expression of GPR78, p-PERK, p-eIF2α, ATF4 and CHOP was significantly reduced in the high and medium dose combination groups (p<0.05). Compared with the groups of Matrine, Osthole and Ribavirin alone groups, the expression of GRP78 was significantly reduced in the high, medium and low dose combination groups (p<0.05), the expressions of p-PERK and CHOP were significantly reduced in the high and medium dose combinatioin groups. The expression of p-eIF2α was significantly reduced in the high dose combination group, compared with the groups of Matrine groups, but no significant difference was found compared with the Osthole group (p<0.05).

In summary, the apoptosis of mice spleen lymphocytes induced by PCV2 were significantly inhibited by the high dose and medium dose of Matrine and Osthole combined treatment through the PERK/eIF2α/ATF4/CHOP pathway activated by GRP78 (p<0.05), although Matrine, Osthole and Ribavirin also have this inhibitory effect.

PCV2 is an important pathogen that causes Post-weaning Multisystemic Wasting Syndrome (PMWS) in weaned piglets, which is also known as PCV2-associated diseases (PCVAD) [2]. It can replicate in multiple tissues of piglets such as liver, kidney, lung, spleen, thymus and lymph nodes, cause interstitial pneumonia and immunosuppression [20]. PCV2 preferentially targets the lymphoid tissues, which leads to immunosuppression of pigs after infection of lymphoid tissue showed a large number of histiocytes and multinucleated giant cell infiltration with decrease and disappearance of lymphocytes [21]. PCV2 can also replicate in the lung, liver, spleen and other tissues of KM mice after infection with PCV2, but it mainly leads to interstitial pneumonia and immunosuppression [22]. Apoptosis of macrophages was reported to be detected in the spleen of mice infected with PCV2 although mice are not the natural hosts of PCV2 [23]. Experiments in laboratory are the gold standard for evaluating the pharmacology of drugs [6]. Therefore, this study evaluated the synergistic anti-PCV2 effect of Matrine combined with Osthole on the basis of establishing PCV2 infected KM mouse model to replace the pig model.

In this study, KM mice infected with PCV2 showed signs of lung interstitial pneumonia, partial lymphocyte loss and macrophage infiltration in the spleen and the expression of Cap gene were higest in the liver. This conclusion is consistent with previous reports [24] in which piglets infected with PCV2 could cause lymphocyte loss and monocytes infiltration, it is also consistent with the Pathological results of spleen HE in PCV2 infected laiwu pigs (LW) and Yorkshire pigs (YL) [25]. Some researchers demonstarated that the typical symptoms of interstitial pneumonia was observed in piglets [26] and KM mice [22] infected with PCV2, our finding is consistent with the reports mentioned above. Yan et al. reported that Matrine and astragalus polysaccharides had a synergisticly protective effect on ulcerative colitis and associated lung injury in rats [27]. It was reported that Matrine combined with Docetaxel had a synergistic effect on prostate cancer [28] and Osthole combined with Cisplatin had synergistic effect on the Rhabdomyosarcoma TE671 Cells of sarcoma [29]. This study revealed that the replication of PCV2 in mouse liver was synergisticly inhibited by the combined use of Matrine and Osthole, which was consistent with the previous results that Matrine combined with Osthole had synergistic anti-PCV2 in vitro [13].

Endoplasmic reticulum (ER) is an important site for virus replication and maturation. Upregulation of molecular chaperone GRP78 is a sign of ER stress. GRP78 can activate the pathways of IRE1, ATF6 and PERK [7], and PCV2 could selectively activate the PERK pathway induced by GRP78, while the IRE1a/XBP1 and ATF6 pathways cannot [8]. ER stress leads PERK to become an active kinase with the ability to phosphorylate α subunits of the eIF2, which induces the preferential translation of ATF4. Then ATF4 activates CHOP to promote apoptosis [30]. Our IHC and Western blot results of the spleen of mice infected with PCV2 at 11 d post infection showed apoptosis of spleen lymphocytes, which was consistent with the previous reaserch finding [23].

In this study, the expression of apoptin was detected in the spleen of mice by Western blot. PCV2 Cap maybe target GPR78, activate PERK apoptosis pathway and induce apoptosis of spleen lymphocytes in PCV2 infected mice. Moreover, the PERK pathway activated by GRP78 was maybe inhibited by the combined use of Matrine and Osthole. The combination treatment inhibited the apoptosis of mouse spleen lymphocytes induced by PCV2, indicating that GRP78 may be a potential target for compound treatment of PCV2 infection in vivo. Gao et al. [31] demonstrated that the expression of GRP78, p-PERK, p-IRE1α and ATF6α in ER pathway was regulated by Matrine and improved ER stress and mitochondrial dysfunction caused by non-alcoholic fatty liver disease. Therefore, GRP78 is an important target for the development of antiapoptotic drugs.

To sum up, Both of Matrine and Osthole have anti-PCV2 effect, and could inhibited PCV2-induced apoptosis by suppressing PERK/eIF2α/ATF4/CHOP/ Bcl-2 pathway mediated by GRP78 (Figure 8). The synergistic inhibitory effect of Matrine combined with Osthole was stronger than that of the two alone. The results provided a new prospect for further investigation of anti-PCV2 infection, and are expected to provide good prescription candidate for the development of novel anti-PCV2 used GRP78 as the therapeutic target.

PCV2, compounds and antibody

PCV2-SH strain was provided by professor Jiang Ping of Nanjing Aricultural University. PK-15 cells were used for virus propagation. The virus titer of PCV2 was 10^6.4TCID₅₀ /mL, which was determined by indirect immunofluorescence assay (IFA).

The chemical structure of Matrine and Osthole was shown in Fig.1. Matrine with 98.7% purity was purchased from National Institutes for Food and Drug Control (lot number:110805-201709). The maximum safe concentration (MSC) used in this experiment was 40 mg/kg [11]. Osthole with 98.0% purity was purchased from Nanjing Zelang Biotechnology Co., Ltd, China (Lot number:ZL20171212SCZS). Osthole was first dissolved in dimethyl sulfoxide (DMSO) and diluted to 12 mg/kg in normal saline, 12 mg/kg of Osthole did not shownhemolysis in hemolysis assay. In this experiment, three dose of Matrine combined with Osthole was used at 40 mg/kg+12 mg/kg (combined high dose), 20 mg/kg+6 mg/kg (combined medium dose) and 10 mg/kg+3 mg/kg (combined low dose ). KM mice were monitored for 14 d after intraperitoneal injection. The mice administrated Matrine combined with Osthole were healthy. Therefore, the combined dose was considered to be safe for KM mice. Ribavirin with 99.0% purity, the positive control drug, was purchased from Beijing Solarbio Technology Co., Ltd, China (CAS: 36791-04-5), the MSC used in this experiment was 40 mg/kg [11].

Antibodies against Cap and p-PERK were purchased from Biorbyt LLC. (California, Britain) and immunology Biology Technology Co., Ltd. (Beijing, China), respectively. GRP78, PERK, eIF2α, p-eIF2α, ATF4, CHOP, Bcl-2, Bax and cleaved caspase-3 were purchased from abcam (Cambridge, USA). GAPDH and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Wuhan Sanying Biology Technology Co., Ltd.（Wu Han, China）and ComWin Biotech Co., Ltd. (Beijing, China) , respectively.

KM mice and administration

All KM mice used in this study was humanely managed according to the animal use protocol approved by the Animal Ethical Committee of the Shanxi Agricultural University. SPF female mice (18-20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal license number is SCXK (Beijing) 2016-0011. Mice were maintained under a 12:12 light/dark cycle with temperature (22±1℃), humidity (60±10%) and receving food and water ad libitum.

One hundred and fifty-four (154) KM were randomly divided into 8 groups after acclimation (Table 1): normal control, PCV2 infected control, combined high dose treatment groups (40 mg/kg+12 mg/kg), combined medium dose treatment groups (20 mg/kg+6 mg/kg), combined low dose treatment groups (10 mg/kg+3 mg/kg), Matrine treatment groups (40 mg/kg), Osthole treatment groups (12 mg/kg), Ribavirin treatment positive control groups (40 mg/kg). 0.5 mL PCV2 with 10^5.4 TCID₅₀/mL was intraperitoneally (i.p.) injected to mice and an equivalent amount of 0.9% saline was i.p. injected to the normal control mice. The compounds was injected i.p. once daily for 5 consecutive days after the mice were infected with PCV2 for 5 days, at a dose of 0.2 mL/10 g, and the control group was given intraperitoneal an equal amount of saline with 10% DMSO at the same frequency and by the same route. Ribavirin was used as the positive drug control group. All mice was weighed daily. The mice were humanely killed at day (d) 5 after PCV2 infection mice (d 0 after compounds adminstration), d 8 after PCV2 infection (d 3 after compounds adminstration), d 11 after PCV2 infection (d 5 after compounds adminstration and without compounds adminstration for 1 day, designated d 6 after compounds adminstration) and d 14 after PCV2 infection (d 5 after compounds adminstration and without compounds adminstration for 4 days, designated as d 9 after compounds adminstration), respectivwlly. The blood was collected into the anticoagulant tube and stored temporarily in a refrigerator at 4℃, the liver and spleen were collected and quickly put into liquid nitrogen for cryopreservation. Blood and tissue samples were stored for further analysis. The lungs and spleen were fixed in 10% neutral buffered formalin for histological analysis. The test groups, the time points of necropsy and the number of mice used are shown in Table 1.

PCR and qPCR analysis

PCV2 DNA was respectively extracted according to the instructions of the blood/cell/tissue DNA extraction kit (TianGen, Beijing, China) from blood, liver and other tissues of PCV2 infected mice d 5. PCV2 DNA concentration was determined by a nucleic acid concentration analyzer (NanoDrop Technologies, Wilmington, DE, USA).

PCR was performed for liver, thymus, spleen, lymph nodes, lung and blood samples collected from mice at 5 dpi. The expression of the Cap gene was detected by polymerase chain reaction (PCR) with primers 5’ TAC ATT TCC AGC AGT TTG and 5’CTC CCG CCA TAC CAT AA. The 148 bp PCR product was analyzed by agarose gel electrophoresisand gel imaging were captured using gel imaging system (Azure Biosystems, Dublin, USA).

The PCV2 DNA of the mice liver was extracted after PCV2 infection at d 5, 8, 11 and 14, respectively and the DNA concentration was measured. The quantitative real-time PCR (qPCR) was applied to amplify the PCV2 Cap gene according to the 2×SYBR Green qPCR Master Mix SYBR (Low ROX, Biotool, USA). Thermal cycling and fluorescence detection were conducted using Applied Biosystems®7500 real-time PCR system. Standard curve of generate by recombinant plasmid vectors containing PCV2 Cap gene fragments was used.

Hematoxylin-eosin（HE）and Immunohistochemical（IHC）

The Bouin’s fixed and paraffin wax embedded lungs and spleens were cut into 4 µm sections. HE staining was performed according to standard protocols and the pathological changes of the lung and spleen tissues were examined.

We performed cleaved caspase-3 immunohistochemistry from paraffin sections to detect cell apoptosis using SP Rabbit&Mouse HRP Kit (DAB, CWBIO, Beijing, China). cleaved caspase-3 monoclonal antibody was incubated for overnight at 4℃. Biotin-labeled secondary antibody working solution and HRP-labeled streptavidin were incubated at room temperature for 10 mins, respectively. Visualization was with diaminobenzoate (DAB) according to standard protocol. The expression of cleaved caspase-3 protein in the spleen tissue was observed by microscope to assess the presence and distribution of the antigen. The equipment used for HE image and IHC image acquisition was（DM3000） (objective lenses: HC PL FLUOTAR 40X/0.8). These micrographs were photographed at a resolution of 10667˟8000 with the cameras of DFC450C, and the image acquisition was obtained using LAS X software, which enhanced the images to 300 dpi. The lamp used for transmitted light BF- contrast imaging was 4W LED illumination.

Measurement of body weight gain rate and viscera index

Body weight of mice in nomal control group and PCV2 infection group were recorded daily. The mice were dissected at d 5 after PCV2 infection and the lung and spleen were weighed to calculate the organ index. These visceras indices were calculated according to the following formula: Viscera index (%) = organ weight (g)/body weight (g) ×100; Body weight gain rate (%) = (body weight before necropsy-body weight before infection) (g)/body weight before infection (g) ×100

Western blot analysis

The total protein was extracted from the liver and spleen of the mice and the concentration was determined using Beyotime Biotechnology kit (Beyotime Biotechnology, Jiangsu, China). The protein samples were separated by SDS-PAGE, then transferred onto the PVDF membrane [13, 32]. The membrane was blocked for 2 h with 5% skim milk and then incubated with the primary antibody at 4°C overnight. The membrane was incubated with the secondary antibody at room temperature for 1.5 h [13]. The proteins levels of PCV2 Cap, GAPDH, GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF4, CHOP, Bcl-2, Bax and cleaved caspase-3 were detected using an eECL Western Blot detection kit (Cwbio Inc, Beijing, China) and chemiluminescence imaging system (BIO-RAD, California, USA), quantified with Image J software (National Institutes of Health, Bethesda, MD, USA) , respectively [13, 32].

Statistical analysis

Data were expressed as the Mean±Standard Errors Mean (SEM) of at least 3 repeated experiments. In this study, differences between the two groups were analyzed using t-test in GraphPad Prism ^TM 5.0 Software (Inc. California, USA), *p˂0.05, ** p˂0.01, ***p˂0.001. “Bonferroni: Compare all pairs of columns” of One-way ANOVA was used for the analysis of difference between multiple groups. The gray intensity of protein bolts was analyzed by Image J software. Different letters (a, b, c, d, etc.) indicated significant difference between groups (p˂0.05).

Ethics approval and consent to participate

All experimental protocols were approved by Animal Ethical Committee of Shanxi Agricultural University, all methods were carried out in accordance with relevant guidelines and regulations. And the study was carried out in compliance with the ARRIVE guidelines.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing financial interests.

Funding

This work was supported by the Shanxi Province Science Foundation for Excellent Youths (Grant No. 201801D211003) , Graduate Education Innovation Project of Shanxi Province (Grant No. 2019BY065) and National Key R&D Program of China (Grant No. 2017YFD0501500). The funders had role in study design, collection, analysis and interpretation of data.

Authors’ contributions

YX, SW, HL and NS designed the research experiments. YX and SW performed the HE experiments, qPCR and Western blot assays. YS, KF, and WY performed the PCR and IHC experiments. YX, PS and NS wrote the manuscript, AK, XZ and JG corrected the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Prof. Jiang (Nanjing Agricultural University) for his assistance in the PCV2.

Authors' information

¹College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi, China.

²School of Public Health, Xinxiang Medical University, Xinxiang 453003, Henan, China

³Laboratory Animal Center, Shanxi Agricultural University, Taigu 030801, Shanxi, China

⁴Department of Veterinary Pathobiology, Schubot Exotic Bird Health Center, Texas A&M University, College Station, Texas, TX 77843, USA

⁵ Medical Research Council (MRC) Centre for Inflammation Research, Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, EH16 4TJ, UK

⁶School of Pharmacy, Heze University, Heze 274000, Shandong, China

Dpi: days post-infection; GRP78: glucose-regulated protein 78; PCV2: porcine circovirus type 2; PERK: protein kinase RNA(PKP)-like extracellular regulated kinase; qPCR: real time quantitative polymerase chain reaction.

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Table 1 The group number of mice used in the experiment and the time points of necropsy

Groups	Normal control	PCV2 infection control	PCV2+ combined high dose	PCV2+ combined medium dose	PCV2+ combined low dose	PCV2 + Matrine	PCV2 + Osthole	PCV2+ Ribavirin control
Groups	Normal control	PCV2 infection control	PCV2+ combined high dose	PCV2+ combined medium dose	PCV2+ combined low dose	PCV2 + Matrine	PCV2 + Osthole	PCV2+ Ribavirin control
Medication d 0	5	5	/	/	/	/	/	/
Medication d 3	6	6	6	6	6	6	6	6
Medication d 6	6	6	6	6	6	6	6	6
Medication d 9	6	6	6	6	6	6	6	6
Total mice	23	23	18	18	18	18	18	18
	154

Additionalfile1.docx
Additional file 1. The original PCR images and HE staining images of Fig. 2, respectively. a The original PCR images of Fig. 2a. b The original HE staining images of Fig. 2b.
Additionalfile2.docx
Additional file 2. Original blot images of Fig. 4d. a-b Original blot images of Cap and GAPDH, respectively.
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Additional file 3. Original HE staining images of mouse lungs in the Fig. 5a. a-h Original HE staining images of Normal group, PCV2 group, High dose mg/kg group, Middle dose group, Low dose group, Matrine group, Osthole group and Ribavirin group, respectively.
Additionalfile4.docx
Additional file 4. Original HE staining images of mouse spleens in the Fig. 5b. a-h Original HE images of Normal group, PCV2 group, High dose mg/kg group, Middle dose group, Low dose group, Matrine group, Osthole group and Ribavirin group, respectively.
Additionalfile5.docx
Additional file 5. Original IHC images of mouse spleens in the Fig. 6a. a-h Normal group, PCV2 group, High dose mg/kg group, Middle dose group, Low dose group, Matrine group, Osthole group and Ribavirin group, respectively.
Additionalfile6.docx
Additional file 6. Original blot images of Fig. 6b. a-d Original blot images of cleaved caspase-3, Bax, Bcl-2, and GAPDH, respectively.
Additionalfile7.docx
Additional file 7. Original blot images of Fig. 7a and 7b. a-d Original blot images of GRP78, p-PERK, t-PERK and GAPDH in the Fig. 7b, respectively. e-i Original blot images of p-eIF2α, t-eIF2α, ATF4, CHOP, Bax and GAPDH in the Fig. 7b, respectively.

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Matrine combined with Osthole inhibited the PERK apoptosis of splenic lymphocytes in PCV2-infected mice model

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