The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2

doi:10.21203/rs.3.rs-29970/v1

Download PDF

Research article

The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2

https://doi.org/10.21203/rs.3.rs-29970/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 12 Oct, 2020

Read the published version in BMC Microbiology →

You are reading this older preprint version

Read the latest preprint version →

Background: Porcine circovirus type 2 (PCV2) is an important DNA pathogen commonly in pig farms, which can cause immunosuppression and induce apoptosis. To evade clearance by the host immune system, PCV2 constantly builds up complex mechanisms or mutates genes, it is difficult to eradicate complex PCV2 infection by relying on vaccine immunity and one compound. At present, there is no literature report on the effective prevention and treatment of PCV2 infection by the combined a of two or more compounds through multiple pathways and multiple targets. We have previously demonstrated the anti-PCV2 effect of Matrine in vitro, but its mechanism has not been further studied. Many literatures have proved that Osthole has a variety of pharmacological activities. Therefore, this study explored the synergistic antiviral effect of Matrine combined with Osthole and the synergistic anti-apoptosis mechanism in the same pathway and targets.

Results: It was first demonstrated that Osthole alone had anti-PCV2 effect and the synergistic anti-PCV2 effect combined with Matrine was better than Matrine and Osthole alone by qPCR, IFA and Western blot results. Subsequently, demonstrated that the reduction of endoplasmic reticulum PERK apoptosis induced by PCV2 was mainly contributed the mechanism of anti-PCV2 using these two extracts by Annexin V-FITC/PI, JC-1 and Western blot results. Finally, it was further demonstrated that Matrine combined with Osthole could inhibit the expression of Cap in cap-transfected PK-15 cells, thus inhibiting Cap-induced PERK apoptosis. In this study, Ribavirin was used as the positive control.

Conclusions: It was proved that the synergistic anti-PCV2 effect of Matrine combined with Osthole was significantly better than that of Matrine and Osthole alone. And the two ombined, with Cap and GRP78 as potential targets of anti-PCV2, further inhibited PERK apoptosis induced by PCV2 infection by directly inhibiting the expression of PCV2 Cap protein. The results formed a basis for further studies on the mechanism of anti-PCV2 in vivo using Matrine combined with Osthole and developing new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets.

General Microbiology

Matrine

Osthole

PCV2

Cap

GRP78

Apoptosis

PCV2 is a main pathogen of porcine circovirus-associated disease with highly infectious and immunosuppression in pig farms [1]. It is the smallest single-stranded cyclic DNA virus with unencapsulated membrane in animals, the capsid protein Cap is a major viral structural protein and a major immunogen involving viral replication of PCV2 [2]. It has been reported that PCV2 could infect mice, piglets and cause disease [3, 4], as well as induce apoptosis [5, 6]. It has caused serious economic losses to the global pig industry for 57 years.

In 2007, the PCV2 vaccine was widely used all over the world [7]. Currently, it is still the main strategy in the prevention and control of porcine circovirus diseases [8, 9]. However, due to the short vaccine protection period and poor cross-protection effect, etc. Vaccine immunity cannot eradicate PCV2 infection [10, 11]. Therefore, the development of effective anti-PCV2 compound has become a hot spot. However, antiviral often constantly builds up complex mechanisms or mutates genes to evade recognition and clearance by the host immune system [12]. PCV2 can cause immunosuppression and it is difficult to effectively control the complex antiviral infection by relying on vaccine immunity and one compound. Therefore, it is necessary to study the combined anti-PCV2 effects of two or more type of compound through multiple targets.

Matrine and Osthole were purchased with a defined plant origin, chemical structure, concentration and specific biological activity. Matrine has anti-inflammatory [13], anti-viral [14, 15], anti-cancer [16], anti-oxidative [17] and anti-apoptotic activities [18]. In our previous studies, Matrine inhibited the proliferation of PCV2 in PK-15 cells, but not studied its mechanism of action [19]. Osthole has a broad prospect of clinical applications in the fields, which has due to its anti-viral [20], anti-inflammatory [21], anti-cancer [22, 23] and anti-apoptotic effects [24]. We speculated that Osthole may has an anti-PCV2 effect.

Therefore, the synergistic antiviral effect and anti-apoptotic mechanism of Matrine combined with Osthole at the targets and pathway were explored in this study. Ribavirin has a broad antiviral activity and is used as the positive control. It provides the theoretical basis for further development of antiviral compounds.

Cytotoxicity of compounds on the PK-15 cells

The cytopathological effect (CPE) was monitored under microscope. Ribavirin was chosen as the positive control. The cells were detached, exhibited round, elongated morphology when they were treated with Osthole and Ribavirin at 0.04 mg/mL and 2 mg/mL (Fig. 2A, the original images see Additional file 1). No obvious change was observed in the cells when 0.01 mg/mL of Osthole was applied, combined with 0.5 mg/mL Matrine and 0.5 mg/mL Ribavirin. The cytopathic rates were 13.68%, 16.45% and 12.37% respectively, which was chosen as the MNTC used for PK-15 cells. The OD value was measured with a microplate reader and the cytopathic ratio was calculated. Charts for CC₅₀ (Figs. 2B and 2C) and CC₅₀ value was generated by GraphPad Prism 5.0. CC₅₀ was 0.02338 ± 0.003331 mg/ mL for Osthole and 0.7119 ± 0.0004273 mg/mL for Ribavirin.

Reproductive Characteristics Of Pcv2

To evaluate the viral load at different time points after 10^4.4 TCID₅₀ PCV2 infected. Cap gene was determined by qPCR. As shown in Fig. 3, th e expression of the Cap gene increased gradually and reached a peak at 48 h. Then, the expression was gradually down-regulated. Therefore, the incubation time point of 48 h after PCV2 infection was adopted in subsequent experiments.

The Synergistic Effects Of Antiviral

To explore the anti-PCV2 effect of compounds, the expression levels of Cap gene was detected by qPCR. The concentrations were designed based on Table 2. The expression of cap gene in the group of Osthole was significantly lower than PCV2 infection group (p < 0.05) (Fig. 4A) and the anti-PCV2 effects of Matrine were verified in our results (Fig. 4B). The synergistic antiviral effects of combined use were investigated by qPCR. The results showed that, compared to PCV2 infection group, the expression of cap gene in the combined treatment group were significantly decreased (p < 0.05). When 0.01 mg/mL of Osthole was combined with Matrine at 0.5, 0.25 and 0.125 mg/mL, respectively, the expression of Cap was significantly lower than the groups treated with 0.5 Matrine and 0.01 Osthole alone (p < 0.05) (Fig. 4C). But, when 0.5 mg/mL of Matrine was combined with 0.01 mg/mL, 0.005 mg/mL and 0.0025 mg/mL of Osthole, no difference on Cap expression was found between them (p > 0.05) (Fig. 4C). The results show that Osthole may plays a major role in the anti-PCV2 effect of combined use. Therefore, 0.01 mg/mL of Osthole combined with 0.5, 0.25 and 0.125 mg/mL of Matrine were used for this experiment.

Table 2

The Final concentration design of different of compounds (mg/mL)
	Matrine	Osthole	Matrine + Osthole	Ribavirin
1	0.5	-	-	-
2	0.25	-	-	-
3	0.125	-	-	-
4	-	0.01	-	-
5	-	0.005	-	-
6	-	0.0025	-	-
7	-	-	0.5 + 0.01	-
8	-	-	0.25 + 0.01	-
9	-	-	0.125 + 0.01	-
10	-	-	0.5 + 0.005	-
11	-	-	0.25 + 0.005	-
12	-	-	0.125 + 0.005	-
13	-	-	0.5 + 0.0025	-
14	-	-	0.25 + 0.0025	-
15	-	-	0.125 + 0.0025	-
16	-	-	-	0.5
Supplementary File list
Additional file. 1 The original microscopic images of Fig. 2A. a-f are Normal cell group, Osthole 0.04 mg/mL, Osthole 0.01 mg/mL, Matrine 0.5 mg/mL + Osthole 0.01 mg/mL, Ribavirin 2 mg/mL and Ribavirin 0.5 mg/mL, respectively.
Additional file. 2 The original IFA images of Fig. 4D. (a) Cell group, (b) Virus group, (c) Matrine 0.5 mg/mL + Osthole 0.01 mg/mL, (d) Matrine 0.25 mg/mL + Osthole 0.01 mg/mL, (e) Matrine 0.125 mg/mL + Osthole 0.01 mg/mL, (f) Matrine 0.5 mg/mL, (g) Osthole 0.01 mg/mL, (h) Ribavirin 0.5 mg/mL.
Additional file. 3 The original blot images of Fig. 4F. (a and b) Original blot images of Cap and GAPDH.
Additional file. 4 The original blot images of Fig. 5C and 5G. (a and b) Original blot images of Cleaved-caspase 3 and GAPDH in the Fig. 5C. (c and g) Original blot images of Cleaved-caspase 3 and GAPDH. (c - g) Original blot image of Cleaved-caspase 9, Bcl-2, Bax, GRP78 and GAPDH in the Fig. 5G, respectively.
Additional file. 5 The original blot images of Fig. 6A. (a - g) Original blot image of p^− PERK, t^− PERK, p^− eIF2α, t^− eIF2α, ATF4, CHOP and GAPDH, respectively.
Additional file. 6 The original blot images of Fig. 7A, 7C and 7H. (a and b) Original blot images of Cap and GAPDH in the Fig. 7A. (C and g) Original blot images of Cap, GRP78, Cleaved-caspase 3, Bcl-2 and GAPDH in the Fig. 7C, respectively. (h - o) Original blot image of p^− PERK, t^− PERK, p^− eIF2α, t^− eIF2α, ATF4, CHOP, Bax and GAPDH in the Fig. 7C, respectively.

The antiviral effects of combined use were also verified by inhibition of expression of Cap protein detected by IFA and Western blot. The results showed that the expression of Cap protein with two compounds was significantly less than use of Matrine or Osthole alone (p < 0.05) (Fig. 4D and 4F). Original fluorescence images of Fig. 4D see Additional file 2 and original blot images of Fig. 4F see Additional file 3.

Inhibition Of Pcv2-induced Apoptosis By Combined Use

To investigate the anti-apoptosis effects of combined use, the samples were stained with apoptosis detection kit and analyzed by flow cytometry. The apoptosis rate in the group treated with combined use was significantly lower than PCV2 infection group (p < 0.05) (Figs. 5A and 5B), the expression of Cleaved-caspase 3 was significantly down-regulated (Figs. 5C-5D) and the mitochondrial membrane potential (MMP) were significantly reduced than PCV2 infection group (p < 0.05) (Figs. 5E and 5F). The expression of Cleaved-caspase9 remained unchanged in the combined concentrations groups of 0.25 + 0.01 and 0.125 + 0.01 than PCV2 control group (p > 0.05). However, those of Cleaved-caspase 3, Bax and GRP78 were significantly down-regulated, the expression of Bcl-2 was significantly elevated (p < 0.05) (Figs. 5G-5K). As shown in Figs. 5E-5K, the down-regulation of MMP did not depend on the caspase family but on the Bcl-2 family. It was speculated that combined use might up-regulate the expression of Bcl-2 in the PERK pathway induced by GRP78, then down-regulate MMP. All the original blot images involved see Additional file 4.

Inhibition of PCV2-induced cell apoptosis by combined use via the ER pathway

The results shown that (Figs. 6A-6E), Compared with the PCV2 infection group, the expressions of p-PERK, p-eIF2α, ATF4 and CHOP in the group treated with Matrine combined with Osthole at different concentrations were significantly decreased (p < 0.05). Indicating combined use could inhibited the PCV2-induced ER apoptosis through the PERK pathway. All the original blot images see Additional file 5.

Intervention of combined use on cell apoptosis through the PERK pathway in the cells transfected with Cap

The expression plasmids carrying the Cap gene were transfected to cells, the expression of Cap protein was detected by Western blot, but not detected in the cells transfected with plasmids without carrying the cap gene (Figs. 7A and 7B). The expression of Cap, GRP78 and Cleaved-caspase 3 in the groups treated with combined use were significantly down-regulated than Cap transfection group (p < 0.05), whereas the expression of Bcl-2 showed a significantly upward trend (p < 0.05) (Figs. 7C-7G). However, the expression of the apoptins p-PERK, p-eIF2α, ATF4, CHOP and Bax in the PERK pathway were significantly elevated than Cap transfection group (p < 0.05) (Figs. 7H-7M). In summary, Matrine combined with Osthole could inhibit cap-induced apoptosis through the PERK pathway. All the original blot images involved see Additional file 6.

We have preliminarily screened and demonstrated the anti-PCV2 effect of Matrine from a variety of compounds, but the anti-PCV2 mechanism of Matrine was unknown [19]. Osthole has anti-virus effect [20]. We confirmed from the level of protein and gene that Matrine combined with Osthole had synergistic anti-PCV2 effect and better than Matrine and Osthole alone. This conclusion provides a theoretical basis for us to study the anti-PCV2 mechanism of multi-target or multi-pathway after Matrine and Osthole compatibility.

Literature has reported that Matrine has anti-apoptotic effect through endoplasmic reticulum pathway and Osthole also has anti-apoptotic effects respectively [18, 25, 26]. In this study, the synergistic inhibitory effect of Matrine combined with Osthole on PCV2-induced apoptosis was demonstrated and the result was consistent with the conclusion that Matrine and Osthole alone have anti-apoptosis effects. PCV2 has been reported to induce mitochondrial apoptosis in PK-15 cells [27, 28]. MMP is recognized as a marker of mitochondrial statement. Our results shown Matrine combined with Osthole decreased MMP. MMP can induce apoptosis by Bcl-2 family, caspase family or by the mitochondrial membrane itself [29]. The proteins in caspase and Bcl-2 families were analyzed by Western blot. Only the expression of Cleaved-caspase 9 in the group treated by 0.5 + 0.01 showed a downward trend and the expression of Cleaved- caspase 3 and Bax were down-regulated and the expression of Bcl-2 was up-regulated. Thus, it is speculated that combined use may down-regulate MMP and inhibit the occurrence of mitochondrial apoptosis by regulating the Bcl-2 and Bax proteins of the Bcl-2 family but the mechanism of inhibiting mitochondrial apoptosis needs more investigation.

ER is an important site for viral replication and maturation. The up-regulation of the molecular chaperone GRP78 is an ER stress marker [30]. GRP78 can activate IRE1, PERK and ATF6 pathways [17]. Zhou et al. [31] reported that PCV2 could activate the PERK pathway and Cleaved-caspase3, Bax and Bcl-2 are also downstream proteins in the PERK apoptosis pathway. To further explore the implication of the changes in Cleaved-caspase3, Bcl-2 and Bax in the ER apoptosis pathway, GRP78 and apoptotic proteins of PERK pathway were analyzed by Western blot. The result shown that the expression of GRP78, p-PERK, p-eIF2a, ATF4 and CHOP showed a downward trend, indicating Matrine combined with Osthole do splayed the anti-PCV2 effect by inhibiting the expression of proteins in the PERK pathway with GRP78 as the target.

Cap is considered to be a major viral structural protein and major immunogen involved in viral replication [2]. In order to further study the effect of Matrine combined with Osthole on PCV2 induced apoptosis by directly inhibiting the expression of PCV2 Cap or by inhibiting apoptosis of the cells to indirectly play the role of anti-PCV2. In this study, cell apoptosis model was established by transfection Cap to simulate PCV2 infected cells. The results of Western blot showed that Matrine combined with Osthole directly inhibited the expression of Cap and thus inhibiting cap-induced apoptosis through the PERK pathway.

In summary, it was proved that the synergistic anti-PCV2 effect of Matrine combined with Osthole was significantly better than that of Matrine and Osthole alone. And the expression of PCV2 Cap protein was directly inhibited by Matrine combined with Osthole and further inhibited the apoptosis induced by PCV2 infection through the PERK/eIF2a/ATF4/CHOP/Bcl-2 pathway using Cap and GRP78 as possible anti-PCV2 targets (Fig. 8). The results provide a theoretical basis for further study on the anti-PCV2 mechanism of multi-target multi-pathway in vivo combined with Matrine and Osthole, the development of new anti-PCV2 compounds targeting Cap and GRP78.

Cells, viruses, compounds and antibodies

The non-PCV-infected pig kidney epithelial cell lines (PK-15) was cultured, passaged, and frozen in liquid nitrogen.

PCV2-SH strain was supplied by Professor Jiang Ping of Nanjing Agricultural University. PK-15 cells were used for virus propagation. 10^6.4 TCID₅₀ / mL of virus titer was determined by indirect immunofluorescence (IFA).

Matrine with 98.7% purity was purchased from the National Institutes for Food and Drug Control (lot number: 110805–201709). Osthole with 98.0% purity was purchased from Nanjing Zelang Biotechnology Co., Ltd (Lot number༚ZL20171212SCZS). Ribavirin with 99.0% purity was purchased from Beijing Solarbio Technology Co., Ltd (CAS༚36791-04-5). The chemical structures of Matrine and Osthole were shown in Fig. 1.

Antibodies against Cap was respectively purchased from Biorbyt LLC. (California, Britain); GRP78、PERK、eIF2ɑ、p-eIF2ɑ、ATF4、Chop、Bcl-2、Bax and Cleaved-caspase 3 were purchased from Abcam (Cambridge, USA); Cleaved-caspase 9 and p-PERK were respectively were purchased from Bioworld Technology Co., Ltd. (Beijing, China) and immunology Biology Technology Co., Ltd. (Beijing, China); 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).

Cytotoxicity Assay

PK-15 cells were seeded onto a 96-well plate at 1 × 10⁶ cells/mL and cultured with Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% fetal calf serum (10% DMEM). When cell confluency reached 80%-90%, the proper concentration of compounds was added. Osthole was dissolved in 1% dimethyl sulfoxide (Solarbio, China), then diluted in DMEM containing 2% fetal calf serum (2% DMEM) and finally prepared eight dilutions for the compounds in 2-fold serial dilutions. Ribavirin (positive control) was dissolved in 2% DMEM, then prepared in serial dilutions. In this laboratory, Sun et al. [27] confirmed that 0.5 mg/mL Matrine has anti-PCV2 replication effect in PK-15 cells. Therefore, in this experiment, the maximum dose of Matrine was set to 0.5 mg/mL. Then, Matrine combined with Osthole were diluted by following Table 1. The equipment used for cytopathic image acquisition is OLYMPUS IX81, which objective lenses is LUCPLANFLN20X/PH1. These micrographs were photographed at a resolution of 4080˟3072 with the cameras of DP71, and the image acquisition was obtained with the software of CELLsens, which enhanced the DPI of the images to 300 dpi. The halogen lamp used for transmitted light Phase contrast imaging is 12V, 100W.

Table 1

The final concentration design of Matrine combined with Osthole
Test number	Matrine(mg/mL)	Osthole(mg/mL)
1	0.5	0.01
2	0.25	0.01
3	0.125	0.01
4	0.5	0.005
5	0.25	0.005
6	0.125	0.005
7	0.5	0.0025
8	0.25	0.0025
9	0.125	0.0025
10	0.5	-
11	0.25	-
12	0.125	-
13	-	0.01
14	-	0.005
15	-	0.0025

Cultured for 60 h, and 100 µL of the fresh DMEM with 10% Cell Counting Kit-8 (Boster, China) was added, then incubated for another hour at 37 °C. The optical density (OD) at 450 nm was measured with a microplate reader (Spectra Max M5,Molecular Devices༌USA). Cytopathic ratio was calculated based on OD value with the formula =[(A-B)/A × 100], in which A and B were the OD value of control and treated cells, respectively. Then, the maximum non-cytotoxic concentration MNTC and CC₅₀ on PK-15 cells were calculated using GraphPad PrismTM5.0 (Inc. California, USA).

Real Time Quantitative Pcr

The PCV2 propagation was determined by real time quantitative PCR (qPCR). When cell confluency reached 80%-90% in the 24-well plate, infect with 10^4.4 TCID₅₀ PCV2 for 2 h. Removed and added 2% DMEM. when the cells were incubated for 6, 12, 24, 48, 72 and 96 h respectively. The copy number of the Cap gene was detected by qPCR. The Primers 5’ TAC ATT TCC AGC AGT TTG and 5’CTC CCG CCA TAC CAT AA were used to amplify PCR product with 148 bp.

The inhibition of PCV2 replication by compounds was also detected by qPCR. The proper concentration of Matrine, Osthole, Matrine combined with Osthole and Ribavirin were added to the cells after PCV2 infected for 2 h. DNA were extracted and

detect Cap gene after incubation for 48 h. The concentrations used were presented in Table 2.

Indirect Immunofluorescence Assay (ifa)

Infect with 10^4.4 TCID₅₀ PCV2 for 2 h. Then Matrine (0.5 mg/mL), Osthole (0.01 mg/mL), Matrine combined with Osthole (0.5 + 0.01, 0.25 + 0.01 and 0.125 + 0.01) and Ribavirin (0.5 mg/mL) were added. The cells were cultured for 48 h at 37 ℃. A mixture of ice-cold acetone and methyl alcohol (1:1) was used to fix the cells for 30 min at -20 °C, washed twice with PBS. PCV2 indirect immunofluorescence kit (China Institute of Veterinary Drug Control) were used to stain the cells. The specific information of IFA image acquisition and processing was the same with the cytopathic test. Besides, The FITC-labeled cells were sequentially excited with a WU excitation block and a WB excitation. Mercury lamp has been used for reflection fluorescence imaging.

Annexin V- Pi Staining For Apoptosis

The different concentrations of Matrine combined with Osthole (high, medium and low) were added after infect with 10^4.4 TCID₅₀ PCV2 for 2 h. The cells were cultured for 48 h, then collected for staining with Annexin V-FITC / PI detection kit (Keygen Biotech, Nanjing, China). The samples were analyzed by flow cytometry (BD Biosciences, USA).

Western Blotting

The anti-PCV2 activity and anti-apoptosis mechanisms of the combined compounds were determined by western blotting. Cells in 6-well plate were infected with 10^4.4 TCID₅₀ PCV2 for 2 h, then treated with compounds for 48 h. The total protein was extracted and determined using the BCA protein concentration detection kit (Beyotime Biotechnology, Jiangsu, China). The protein samples were separated by SDS-PAGE, then transferred to the PVDF membrane. The membrane was blocked for 2 h with 5% skim milk, then incubated with the primary antibody at 4 °C overnight. After being washed three times with TBST, the membrane was incubated with the secondary antibody at room temperature for 1.5 h then was detected using an eECL Western Blot detection kit (Cwbio, Beijing, China) and chemiluminescence imaging system (BIO-RAD, California, USA).

Effects of the combined drugs on MMP detected by JC-1

Cells in 6-well plate were incubated with 10^4.4 TCID₅₀ PCV2 for 2 h, then treated with compounds for 48 h. The harvested cells were treated with JC-1 mitochondrial membrane potential (MMP) detection kit (Beyotime Biotechnology, Jiangsu, China), analyzed by flow cytometry.

Cap recombinant plasmid construction, cell transfection and its mechanism on apoptosis analyzed by Western blot

The optimizing Cap gene sequence was inserted into plasmid pCDNA3.1 at the KpnI and XhoI cutting sites. The plasmid was transformed into the E coli. The bacteria with right Cap sequence was used for the plasmid extraction with GoldHi EndoFree Plasmid Maxi Kit (CW2104M, Cwbio, China). The plasmid concentration was determined by a biological mass spectrometer (D30, Eppendorf, Germany).

When cell confluency in 6-well plate reached 70%-90%, the pCDNA3.1-cap (p-cap) and pCDNA3.1-vector (p-vector) alone were transfected using 7.5 µL Lipofectamine 2000(Invitrogen, California, USA) respectively and 2.5 µg of p-cap were mixed with 125 µL of Opti-MEM. The mixture was incubated for 15 min at room temperature. 50 µL of the mixture was added dropwise to the cells and 6 h incubation. Matrine combined with Osthole were added and cultured for 48 h. p-vector and Ribavirin as the control. Collected and analyzed the protein expression levels by western blot for Cap protein and the key apoptin of PERK pathway.

Statistical analysis

CC₅₀ was calculated using nonlinear regression, and the results of “log (inhibitor) vs. response-variable slope” and the data of generated by qPCR, IFA, Western blot, Annexin V-FITC/PI and JC-1 were analyzed by GraphPad Prism TM 5.0. Image J (National Institutes of Health, USA) was used to measure the gray intensity of protein bolts. All data are expressed as the mean ± standard error of the mean (SEM). Statistical significance threshold was set at p < 0.05.

ATF4 activating transcription factor 4; Bax:BCL2-associated X protein; Bcl-2:B-cell lymphoma-2; CHOP:C/EBP-homologous protein; eIF2α:eukaryotic translation initiation factor 2a; GRP78:glucose-regulated protein 78; MNTC:maximum non-cytotoxic concentration; PCV2:porcine circovirus type 2; PERK:protein kinase RNA(PKP)-like extracellular regulated kinase; RT-PCR:real time quantitative polymerase chain reaction.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and the datasets are provided from the corresponding author on reasonable request.

Competing interests

All authors declared no competing conflict of interest.

Funding

This work was supported by the National Key R&D Program of China (Grant No. 2017YFD0501500), Shanxi Province Science Foundation for Excellent Youths (Grant No. 201801D211003) and Graduate Education Innovation Project of Shanxi Province (Grant No. 2019BY065). Financial support personnel all have a certain role in the design all experiments and approved the final manuscript.

Authors' contributions

YX, NS and HL designed all the experiments. YX, PS and SW performed the cell culture experiments, qPCR and Immunofluorescence assay. YS, KF and WY performed the Western blot assay and Cap transfection assay. YX, JG and XZ wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

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

Author' information

¹ College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, Shanxi, China

Tischer I, Gelderblom H, Vettermann W, Koch MA. A very small porcine virus with circular single-stranded DNA. Nature. 1982;295(5844):64–6.
Nawagitgul P, Morozov I, Bolin SR, Harms FA, Sorden SD, Paul PS. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein. J GenVirol. 2000;81(9):2281–7.
Kiupel M, Stevenson GW, Galbreath EJ, North A, HogenEsch H, Mittal SK. Porcine circovirus type 2 (PCV2) causes apoptosis in experimentally inoculated BALB/c mice. BMC Vet Res. 2005;1(1):1–7.
Sinha A, Schalk S, Lager KM, Wang C, Opriessnig T. Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs. Res Vet Sci. 2012;92(1):151–6.
Wei L, Zhu Z, Wang J, Zhang C, Quan R, Yan X. Regulatory role of ASK1 in porcine circovirus type 2-induced apoptosis. Virology. 2013;447(1–2):285–91.
Zhang Y, Sun R, Geng S, Shan Y, Li X. Porcine circovirus type 2 induces ORF3-independent mitochondrial apoptosis via perk activation and elevation of cytosolic calcium. J Virol. 2019;93(7):01784–800.
Chae C. Commercial porcine circovirus type 2 vaccines: efficacy clinical application. Vet J. 2012;194(2):151–7.
Franzo G, Tucciarone CM, Cecchinato M, Drigio M. Porcine circovirus type 2 (PCV2) evolution before and after the vaccination introduction: A large scale epidemiological study. Sci Rep. 2016;6:39458–70.
Xiao CT, Halbur PG, Opriessnig T. Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d. J Gen Virol. 2015;96:1830–41.
Beach NM, Meng XJ. Efficacy and future prospects of commercially available and experimental vaccines against porcine circovirus type 2 (PCV2). Virus Res. 2012;164(1–2):33–42.
Zhai SL, Chen SN, Xu ZH, Tang MH, Wang FG, Li XJ, Sun BB, Deng SF, Hu J, Lv DH, Wen XH, Yuan J, Luo ML, Wei WK. Porcine circovirus type 2 in China: an update on and insights to its prevalaence and control. Virol J. 2014;11:88–99.
Li T, Peng T. Traditional Chinese herbal medicine as a source of molecules with antiviral activity. Antivir Res. 2013;97(1):1–9.
Niu YJ, Dong QM, Li RH. Matrine regulates Th1/Th2 cytokine responses in rheumatoid arthritis by attenuating the NF-κB signaling. Cell Biol Int. 2017;41(6):611–21.
Sun N, Wang ZW, Wu CH, Li E, He JP, Wang SY, Hu YL, Lei HM, Li HQ. Antiviral ctivity and underlying molecular mechanisms of Matrine against porcine reproductive and respiratory syndrome virus in vitro. Res Vet Sci. 2014;96(2):323–7.
Gao X, Guo S, Zhang S, Liu A, Shi L, Zhang Y. Matrine attenuates endoplasmic reticulum stress and mitochondrion dysfunction in nonalcoholic fatty liver disease by regulating SERCA pathway. J Transl Med. 2018;16(1):319–35.
Liou CJ, Lai YR, Chen YL, Chang YH, Li ZY, Huang WC. Matrine Attenuates COX-2 and ICAM-1 Expressions in Human Lung Epithelial Cells and Prevents Acute Lung Injury in LPS-Induced Mice. Mediat Inflamm. 2016; 2016:1–12.
Zhao P, Zhou R, Zhu XY, Hao YJ, Li N, Wang J, Niu Y, Sun T, Li YX, Yu JQ. Matrine attenuates focal cerebral ischemic injury by improving antioxidant activity and inhibiting apoptosis in mice. Int J Mol Med. 2015;36(3):633–44.
Chang JL, Hu SP, Wang WY, Li YM, Zhi WL, lu S, Shi Q, Wang YJ, Yang YP. Matrine inhibits prostate cancer via activation of the unfolded protein response/endoplasmic reticulum stress signaling and reversal of epithelial to mesenchymal transition. Mol Med Rep. 2018;18(1):945–57.
Sun N, Yu T, Zhao JX, Sun YG, Jiang JB, Duan ZB, Wang WK, Hu YL, Lei HM, Li HQ. Antiviral activities of natural compounds derived from traditional Chinese medicines against porcine circovirus type 2 (PCV2). Biotechnol Bioproc E. 2015;20(1):180–7.
Huang RL, Chen CC, Huang YL, Hsieh DJ, Hu CP, Chen CF, Chang C. Osthole increases glycosylation of hepatitis B surface antigen and suppresses the secretion of hepatitis B virus in vitro. Hepatology. 1996;24(3):508–15.
Huang WC, Liao PC, Huang CH, Hu S, Huang SC, Wu SJ. Osthole attenuates lipid accumulation, regulates the expression of inflammatory mediators, and increases antioxidants in FL83B cells. Biomed Pharmacother. 2017;91:78–87.
Lin VC, Chou CH, Lin YC, Lin JN, Yu CC, Tang CH, Lin HY, Way TD. Osthole suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt/mTOR pathway. J Agric Food Chem. 2010;58(8):4786–93.
Liu PY, Chang DC, Lo YS, His YT, Lin CC, Chuang YC, Lin SH, Hsieh MJ, Chen MK. Osthole induces human nasopharyngeal cancer cells apoptosis through Fas-Fas ligand and mitochondrial pathway. Environ Toxicol. 2018;33(4):446–53.
Li K, Ding D, Zhang M. Neuroprotection of Osthole against Cerebral Ischemia/Reperfusion Injury through an Anti-apoptotic Pathway in Rats. Biol Pharm Bull. 2016;39(3):336–42.
Xiao Y, Ma DC, Wang HL, Wu DM, Chen Y, Ji K, Qin T, Wu L. Matrine Suppresses the ER-positive MCF Cells by Regulating Energy Metabolism and Endoplasmic Reticulum Stress Signaling Pathway. Phytother Res. 2017;31(4):671–9.
Ji HJ, Hu JF, Wang YH, Chen XY, Zhou R, Chen NH. Osthole improves chronic cerebral hypoperfusion induced cognitive deficits and neuronal damage in hippocampus. Eur J Pharmacol. 2010;636(1–3):96–101.
Lin C, Gu JY, Wang HJ, Zhou JW, Li JR, Wang SN, JinYL, Liu CM, Liu J, Yang HC, Jiang P, Zhou JY. Caspase-dependent apoptosis induction via viral protein ORF4 of porcine circovirus 2 binding to mitochondrial adenine nucleotide translocase 3. J Virol. 2018;92(10):e00238–256.
Chen HL, Yang J, Fu YF, Meng XN, Zhao WD, Hu TJ. Effect of total flavonoids of Spatholobus suberectus Dunn on PCV2 induced oxidative stress in RAW 264.7 cells. BMC Complement Altern M. 2017;17(1):244–53.
Adeyemi RO, Landry S, Davis ME, Weitzman MD, Pintel DJ. Parvovirus minute virus of mice induces a DNA damage response that facilitates viral replication. Plos Pathog. 2010;6(10):e1001141-1152.
Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Bio. 2012;13(2):89–102.
Zhou YS, Qi BZ, Gu YX, Xu F, Du HH, Li XL, Fang WH. Porcine circovirus 2 deploys PERK pathway and GRP78 for its enhanced replication in PK-15 cells. Viruses. 2016;8(2):56–72.

Additional file. 1 The original microscopic images of Fig. 2A. a-f are Normal cell group, Osthole 0.04 mg/mL, Osthole 0.01 mg/mL, Matrine 0.5 mg/mL + Osthole 0.01 mg/mL, Ribavirin 2 mg/mL and Ribavirin 0.5 mg/mL, respectively.

Additional file. 2 The original IFA images of Fig. 4D. (a) Cell group, (b) Virus group, (c) Matrine 0.5 mg/mL + Osthole 0.01 mg/mL, (d) Matrine 0.25 mg/mL + Osthole 0.01 mg/mL, (e) Matrine 0.125 mg/mL + Osthole 0.01 mg/mL, (f) Matrine 0.5 mg/mL, (g) Osthole 0.01 mg/mL, (h) Ribavirin 0.5 mg/mL.

Additional file. 3 The original blot images of Fig. 4F. (a and b) Original blot images of Cap and GAPDH.

Additional file. 4 The original blot images of Fig. 5C and 5G. (a and b) Original blot images of Cleaved-caspase 3 and GAPDH in the Fig. 5C. (c and g) Original blot images of Cleaved-caspase 3 and GAPDH. (c - g) Original blot image of Cleaved-caspase 9, Bcl-2, Bax, GRP78 and GAPDH in the Fig. 5G, respectively.

Additional file. 5 The original blot images of Fig. 6A. (a - g) Original blot image of p^-PERK, t^-PERK, p^-eIF2^α, t^-eIF2^α, ATF4, CHOP and GAPDH, respectively.

Additional file. 6 The original blot images of Fig. 7A, 7C and 7H. (a and b) Original blot images of Cap and GAPDH in the Fig. 7A. (C and g) Original blot images of Cap, GRP78, Cleaved-caspase 3, Bcl-2 and GAPDH in the Fig. 7C, respectively. (h - o) Original blot image of p^-PERK, t^-PERK, p^-eIF2^α, t^-eIF2^α, ATF4, CHOP, Bax and GAPDH in the Fig. 7C, respectively.

Download PDF

Journal Publication

published 12 Oct, 2020

Read the published version in BMC Microbiology →

Editorial decision: Major revision
30 Jul, 2020
Review #2 received at journal
27 Jul, 2020
Review #1 received at journal
15 Jul, 2020
Reviewer #2 agreed at journal
09 Jul, 2020
Reviewer #1 agreed at journal
08 Jul, 2020
Reviewers invited by journal
06 Jul, 2020
Editor assigned by journal
16 Jun, 2020
Submission checks completed at journal
15 Jun, 2020
Editor invited by journal
15 Jun, 2020

You are reading this older preprint version

Read the latest preprint version →

The combined usage of Matrine and Osthole inhibited endoplasmic reticulum apoptosis induced by PCV2

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Discussion

Conclusion

Methods

Cells, viruses, compounds and antibodies

Cytotoxicity Assay

Real Time Quantitative Pcr

Indirect Immunofluorescence Assay (ifa)

Annexin V- Pi Staining For Apoptosis

Western Blotting

Statistical analysis

Abbreviations

Declarations

Ethics approval and consent to participate

Funding

Authors' contributions

Acknowledgements

References

Supplementary Materials

Supplementary Files

Status:

Journal Publication

Version 1