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. 2020 Oct 12;20(1):303.
doi: 10.1186/s12866-020-01986-2.

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

Yinlan Xu  1 , Panpan Sun  2 , Shuangxiu Wan  1 , Jianhua Guo  3 , Xiaozhong Zheng  4 , Yaogui Sun  1 , Kuohai Fan  2 , Wei Yin  1 , Na Sun  1 , Hongquan Li  5
Affiliations

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

Yinlan Xu et al. BMC Microbiol. .

Abstract

Background: Porcine circovirus type 2 (PCV2) is an important and common DNA virus that infect pig and can cause immunosuppression and induce apoptosis in the infected cells. To escape the host immune system, PCV2 constantly builds up complex mechanisms or mutates genes, and that is why it is difficult to eradicate complex PCV2 infection by relying on vaccines and single compound. At present, there is few literature reports on the effective prevention and treatment of PCV2 infection by a combination of two or more compounds. Previously, we have demonstrated the anti-PCV2 effect of Matrine in vitro, but its mechanism has not been further evaluated. Literatures have proven that Osthole has a variety of pharmacological activities, and we tested the ability of Osthole to inhibit PCV2 replication in cell culture. Therefore, this study explored the synergistic antiviral effect of Matrine combined with Osthole and their synergistic anti-apoptotic mechanism.

Results: Osthole alone had an anti-PCV2 effect, and then its synergistic anti-PCV2 effect of Osthole and Matrine was better than that of Matrine or Osthole alone as demonstrated by qRT-PCR, IFA and Western blotting results. The anti-apoptotic mechanism of these two compounds by inducing the PERK pathway by PCV2 was elucidated through Annexin V-FITC/PI, JC-1 and Western blotting. Matrine and Osthole combination could inhibit the expression of Cap in Cap-transfected PK-15 cells, thus inhibiting Cap-induced PERK apoptosis. Ribavirin was used as a positive control.

Conclusions: The combination of Osthole and Matrine had the synergistic effect of anti-PCV2 infection by directly inhibiting the expression of PCV2 Cap protein. The combination of these two compounds also inhibited PERK apoptosis induced by PCV2 Cap protein, possibly by regulating the level of GRP78. The results formed a base for further studies on the mechanism of anti-PCV2 in vivo using Matrine and Osthole combination and developing new anti-PCV2 compounds with Cap and GRP78 as therapeutic targets.

Keywords: Apoptosis; Cap; GRP78; Matrine; Osthole; PCV2.

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Conflict of interest statement

All authors declared no competing conflict of interest.

Figures

Fig. 1
Fig. 1
Cytotoxicity of compounds on PK-15 cells detected by CCK8. When the monolayer of PK-15 cells was formed, the compounds were added to the cells and then incubated for 60 h. The morphological changes were observed in the wells treated with Osthole, Matrine combined with Osthole, and Ribavirin. According to the experimental design, the composite image is the product of two time points. a when a low concentration (0.01 mg/mL Osthole, 0.5 mg/mL Matrine + 0.01 mg/mL Osthole, and 0.5 mg/mL Ribavirin) was applied, cells were highly refractive and healthy. b and c CC50 curves of Osthole and Ribavirin. The direct correlation of the cytopathic rate with Osthole and Ribavirin is shown. Clearly indicating that the cytotoxicity increased with a higher concentration of Osthole or Ribavirin
Fig. 2
Fig. 2
Replication of PCV2 in PK-15 cells. PK-15 cells were infected with 104.4 TCID50 of PCV2. Virus copy numbers were detected by qRT-PCR from the samples collected at 6, 12, 24, 48, 72 and 96 h after the initial infection. The chart plotted with virus copy number against the time points after the infection was generated, which showed that the virus replication reached a plateau at 48 h after infection. The virus number was very low in the first 24 h of infection and then dramatically increased between 24 h and 48 h after infection, reaching a plateau at 48 h. Therefore, all samples were collected at 48 h after infection to analyze the anti-viral effect and anti-apoptotic mechanisms of the compounds
Fig. 3
Fig. 3
Anti-PCV2 activities of Matrine combined with Osthole detected by qRT-PCR, IFA and Western blotting. a-c Expression of the Cap gene was detected by qRT-PCR in cells treated with compounds; d and e Expression of Cap protein was detected by IFA; e and g Expression of Cap was detected by Western blotting and ImageJ was used for the quantification of cap protein bands in Western blotting. GraphPad Prism™ 5.0 was used for the statistical analysis. Data are expressed in the form of mean ± SEM, with different letters (a, b, c, d, etc.) indicating a significant difference from other groups (p < 0.05)
Fig. 4
Fig. 4
PCV2-induced PK-15 cell apoptosis inhibited by Matrine combined with Osthole. After the PK-15 cells were infected with 104.4 TCID50 of PCV2, the cell samples were harvested after 48 h incubation with Matrine combined with Osthole. a and b The apoptotic rate was analyzed by flow cytometry. Data indicate the sum of the late apoptosis of the right upper quadrant and the early apoptosis of the right lower quadrant. c and d The level of cleaved caspase-3 was down-regulated significantly in the Western blotting. e and f Changes in MMP after treatment for 48 h with a combination of drugs were analyzed by JC-1. R2 represents the changes in the MMP. g-k Expression of the key apoptins were analyzed by Western blotting and grayscale analysis. Data are expressed in the form of mean ± SEM, with different letters (a, ,b, c, d, etc.) indicating a significant difference from other groups (p < 0.05)
Fig. 5
Fig. 5
Inhibition of PCV2-induced cell apoptosis by Matrine combined with Osthole via the ER pathway. After the PK-15 cells were infected with104.4 TCID50 of PCV2 for 2 h, cells were treated with Matrine and Osthole combination for 48 h, and cell samples were collected. a-e The expression levels of each apoptin in the PERK pathway were analyzed. After treatment with Matrine and Osthole combination, the levels of p-PERK, p-eIF2α, ATF4 and CHOP were down-regulated. Data are expressed in the form of mean ± SEM, with different letters (a, b, c, d, etc.) indicating a significant difference from other groups (p < 0.05)
Fig. 6
Fig. 6
Matrine combined with Osthole inhibits via the PERK pathway apoptosis in the cells transfected with Cap. After the PK-15 cells were transfected with p-Cap for 6 h, the cells were treated with Matrine and Osthole combination for 48 h and the samples were harvested. a and b The level of Cap protein in the group transfected with p-Cap was markedly elevated in the Western blotting. c-g As revealed by Western blotting, Matrine combined with Osthole down-regulated the marker protein GRP78 in the ER pathway, as well as the expression levels of the cleaved caspase-3 and Bcl-2 in the PERK apoptosis pathway. h-o Levels of apoptins in the PERK pathway were determined by Western blotting. Data are expressed in the form of mean ± SEM, with different letters (a, b, c, d, etc.) indicating a significant difference from other groups (p < 0.05)
Fig. 7
Fig. 7
Mechanism of anti-apoptosis induced by PCV2 or Cap using Matrine combined with Osthole through by ER pathway. The ‘arrow’ indicates a promoting effect, whereas red ‘T-shape’ indicates an inhibiting effect. Virus replication is inhibited by inhibiting the expression of apoptins in the PERK pathway of ER, further down-regulating the expression of downstream pro-apoptins cleaved caspase-3 and Bax, and up-regulating the expression of anti-apoptins Bcl-2
Fig. 8
Fig. 8
Chemical structure of Matrine and Osthole
See this image and copyright information in PMC

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