Abstract:

Objective To discuss the effect of parthenolide （PTL） on the apoptosis of the chondrocytes under mechanical stretch stress by regulating the expression of piezo type mechanosensitive ion channel component 1（Piezo1）， and to clarify the related mechanism. Methods The chondrocytes were divided into 0%， 5%， 10%， 15%， and 20% stretch groups according to the stretch variable. Additionally， the chondrocytes were divided into control group， 20% stretch group， 20% stretch+5 μmol·L^-1 PTL group， 20% stretch+10 μmol·L^-1 PTL group， and 20% stretch+20 μmol·L^-1 PTL group. The Piezo1 short hairpin RNA （shRNA） interference lentivirus （sh-Piezo1） or shRNA-NC lentivirus were used to infect the chondrocytes， and the chondrocytes were divided into sh-Piezo1 group and sh-NC group， and also set up blank control group. The chondrocytes were also devided into 20% stretch group， 20% stretch+PTL group， 20% stretch+sh-Piezo1 group， and 20% stretch+sh-Piezo1+PTL group. Hoechst 33258 fluorescence staining was used to observe the morphology of the nuclear in various groups； flow cytometry was used to detect the apoptotic rates of the cells in various groups； spectrophotometry was used to detect the cysteinyl aspartate specific proteinase（Caspase）-3 activities in the cells in various groups； CCK-8 method was used to detect the proliferation rates of the cells in various groups； Fluo-4/AM fluorescent probe method was used to detect the calicium ion（Ca²⁺） levels in the cells in various groups； real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of Piezo1 mRNA in the cells in various groups； Western blotting method was used to detect the expression levels of Piezo1 protein in the cells in various groups. Results The Hoechst 33258 fluorescence staining resuts showed that as the increasing of stretch， the number of the chondrocytes with fragmented and densely stained nuclei in 0%， 5%， 10%， 15%， and 20% stretch groups were gradually increased. The flow cytometry results showed that compared with 0% stretch group， the apoptotic rates of the chondrocytes in 5%， 10%， 15%， and 20% stretch groups were significantly increased （P<0.01）； compared with control group， the apoptotic rate of the chondrocytes in 20% stretch group was significantly increased （P<0.05）； compared with 20% stretch group， the apoptotic rates of the chondrocytes in 20% stretch+ 5 μmol·L^-1 PTL group， 20% stretch+10 μmol·L^-1 PTL group， and 20% stretch+20 μmol·L^-1 PTL group were significantly decreased （P<0.05）； compared with 20% stretch group， the apoptotic rates of chondrocytes in 20% stretch+PTL group and 20% stretch+sh-Piezo1 group were significantly decreased （P<0.05）. The spectrophotometry results showed that compared with 0% stretch group， the Caspase-3 activities in the chondrocytes in 5%， 10%， 15%， and 20% stretch groups were significantly increased （P<0.01）； compared with control group， the Caspase-3 activity in the chondrocytes in 20% stretch group was significantly increased （P<0.05）； compared with 20% stretch group， the Caspase-3 activities in the chondrocytes in 20% stretch+5 μmol·L^-1 PTL group， 20% stretch+10 μmol·L^-1 PTL group， and 20% stretch+20 μmol·L^-1 PTL group were significantly decreased （P<0.05）. Compared with 20% stretch group， the Caspase-3 activities in the chondrocytes in 20% stretch+PTL group and 20% stretch+ sh-Piezo1 group were significantly decreased （P<0.05）. The CCK-8 method results showed that compared with 0 μmol·L^-1 PTL group， the proliferation rates of the chondrocytes in 40.00， 80.00， and 160.00 μmol·L^-1 PTL groups were significantly decreased （P<0.05）， indicating that 20.00 μmol·L^-1 PTL was the maximum non-toxic concentration. The Fluo-4/AM fluorescent probe method results showed that compared with control group， the Ca²⁺ level in the chondrocytes in 20% stretch group was significantly increased （P<0.05）； compared with 20% stretch group， the Ca²⁺ levels in the chondrocytes in 20% stretch+5 μmol·L^-1 PTL group， 20% stretch+10 μmol·L^-1 PTL group， and 20% stretch+ 20 μmol·L^-1 PTL group were significantly decreased （P<0.05）； compared with 20% stretch group， the Ca²⁺ levels in the chondrocytes in 20% stretch+PTL group and 20% stretch+sh-Piezo1 group were significantly decreased （P<0.05）. The RT-qPCR results showed that compared with blank control group and sh-NC group， the expression level of Piezo1 mRNA in the chondrocytes in sh-Piezo1 group was significantly decreased （P<0.05）. The Western blotting results showed that compared with control group， the expression levels of Piezo1 protein in the chondrocytes in 20% stretch group was significantly increased （P<0.05）； compared with 20% stretch group， the expression levels of Piezo1 protein in the chondrocytes in 20% stretch+5 μmol·L^-1 PTL group， 20% stretch+10 μmol·L^-1 PTL group， and 20% stretch+20 μmol·L^-1 PTL group were significantly decreased （P<0.05）； compared with blank control group and sh-NC group， the expression level of Piezo1 protein in the chondrocytes in sh-Piezo1 group was significantly decreased （P<0.05）. Conclusion PTL can inhibit the apoptosis of the chondrocyte induced by high-intensity cyclic mechanical stretch stress， and its mechanism may be related to inhibiting the Piezo1-mediated Ca²⁺ influx-induced apoptosis.

Key words: Parthenolide, Piezo type mechanosensitive ion channel component 1, Mechanical stretch stress, Chondrocytes, Apoptosis