Neuroprotective effect and mechanism of dendrobium alkaloids on Ht22 cell injury induced by OGD/R

Abstract:

Objective：To investigate the neuroprotective effect and mechanism of dendrobium alkaloids（DNLA） on the Ht22 cell injury induced by oxygen glucose deprivation/reperfusion（OGD/R）. Methods：Ht22 cell lines were cultured in vitro and randomly divided into 9 groups，i.e. control group（without any treatment），OGD/R group，OGD/R+ low-dose DNLA treatment group（0.03 mg/mL），OGD/R + medium-dose DNLA treatment group（0.3 mg/mL），OGD/R + high-dose DNLA treatment group（3 mg/mL），OGD/R+VX765（inhibitor of Caspase-1） group（10 ng/mL），OGD/R+PEG3000→Media group（normal medium was supplemented with medium containing PEG3000（1%） in the process of OGD/R），OGD/R+Media→PEG3000 group（normal medium was supplemented with medium containing PEG3000（1%） after the process of OGD/R） and PEG3000（1%） group. OGD/R model was established for Ht22 cells after a 2 h oxygen glucose deprivation and 24 h reperfusion. DNLA was added from 12 hours before oxygen glucose deprivation and continued to the end of reperfusion. VX765 was added from the beginning of oxygen glucose deprivation and continued to the end of reperfusion. Cell activity rate was de－tected by MTT assay，and cell injury rate was determined by LDH assay. Then OGD/R+high-dose DNLA treatment group（3 mg/mL） was chosen for subsequent tests. Cell pyroptotic rate was measured by flow cytometry Annexin V FIFT/PI double staining，and expressions of pyroptosis-related proteins Caspase-1，GSDMD and GSDMD-C，were detected by Western blot assay，as well as in－terleukin-1β（IL-1β），interleukin-6（IL-6） and interleukin-18（IL-18）. The expression of Caspase-1 in Ht22 cells was detected by immunofluorescence staining，and the process of pyroptotic Ht22 cells was observed by scanning electron microscopy（SEM）. Results：The pyroptotic process of Ht22 cells was observed by SEM after OGD/R. Compared with the control group，cell activity notably decreased in the OGD/R group（P=0.008），while cell injury rate and pyroptotic rate increased（P=0.009，P=0.005）. Expression of Caspase-1（P=0.000） and GSDMD（P=0.001） proteins were significantly increased，while expression of GSDMD-C（P=0.004） interest－ingly decreased. The protein expression levels of inflammatory cytokines IL-1β（P=0.006），IL-6（P=0.004） and IL-18（P=0.000） were also significantly increased，and the viability and injury rate of Ht22 cells in the PEG3000 group were not obviously changed（P=0.005，P=0.007）. Compared with the OGD/R group，the 3 DNLA treatment groups showed increased cell viability and signif－icantly decreased cell injury rate and pyroptotic rate，especially in the high-dose DNLA treatment group（P=0.005，P=0.009，P=0.003）. The expression of Caspase-1，GSDMD that related to pyroptosis decreased a lot（P=0.005，P=0.008），while GSDMD-C proteins levels increased a lot（P=0.002），and the protein expression levels of inflammatory cytokines IL-1β（P=0.005），IL-6（P=0.000） and IL-18（P=0.007） were decreased. The cell viability also increased in the OGD/R+VX765 group（P=0.007），and at the same time，the cell injury rate and pyroptotic rate obviously decreased（P=0.002，P=0.008）. The expression levels of Caspase-1（P=0.006） and GSD－MD（P=0.007） were decreased，while the expression levels of GSDMD-C（P=0.004） increased. The expression levels of inflammatory cytokines，IL-1β（P=0.006），IL-6（P=0.004） and IL-18（P=0.001），were significantly increased. Intriguingly，compared with the OGD/R+Media→PEG3000 group，the cell viability rate of the OGD/R+PEG3000→Media group increased（P=0.007，P=0.006）and its cell injury rate decreased（P=0.006，P=0.003）. Conclusions：The Ht22 cells induced by OGD/R showed the process of pyroptosis. DNLA has a neuroprotective effect on OGD/R-induced Ht22 cell damage. The mechanisms may involve with the inhibition of the expression of Caspase-1，a protein related to pyroptosis.