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

Abstract

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.

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

“…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].…”

“…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 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), quanti ed with Image J software (National Institutes of Health, Bethesda, MD, USA) , respectively [13,32].…”

“…Matrine is an alkaloids and the main bioactive component of sophora. It has anti-in ammatory [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-in ammatory [15], anti-cancer [16], anti-virus [17], anti-immunosuppression [18] and anti-apoptosis effects [19].…”

“…Osthole is the main bioactive ingredient of Fructus Cnidii and has anti-in ammatory [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.…”