Objective To investigate the effect of Yes-associated protein(YAP)on intervertebral disc nucleus pulposus cells and its possible mechanism.Methods The relatively normal and degenerative intervertebral disc tissues of patients who underwent lumbar surgery in our hospital from March 2021 to July 2022 were harvested,and then the expression of YAP in the tissues were detected by immunohistochemistry assay and Western blotting.Human primary nucleus pulposus cells were isolated and primarily cultured,and treated with IL-1β to induce degeneration.Then the cells was divided into control group,IL-1β group,IL-1β+LV-YAP group,IL-1β+YAP-siRNA group,and IL-1β+LV-YAP+3-MA group.Western blot analysis was used to detect the expression of the proteins related to extracellular matrix catabolism and autophagy in each group.Finally,a rat model of disc degeneration was established,and the expression of YAP and LC3 and disc degeneration were observed with MRI,Alcian blue staining and immunohistochemistry.Results The expression level of YAP was significantly lower in the degraded disc tissues than the relatively normal disc tissues(P＜0.05).The IL-1β+LV-YAP group had significantly increased protein levels of Collagen Ⅱ,Aggrecan,and LC3-11(P＜0.05),and decreased levels of MMP-3 and MMP-13(P＜0.05)when compared with the cells after IL-1β treatment,whereas the IL-1β+YAP-siRNA group showed the exact opposite effects.What's more,pretreatment with autophagy inhibitor 3-MA resulted in decreased number of GFP-LC3 positive particles and protein levels of Collagen Ⅱ,Aggrecan and LC3-Ⅱ(P＜0.05),and increased protein expression of MMP-3 and MMP-13(P＜0.05)in comparison with the conditions in the IL-1β+LV-YAP group.Furthermore,YAP overexpression promoted LC3 expression and inhibited disc degeneration in rat model of disc degeneration.Conclusion YAP overexpression can inhibit extracellular matrix degradation by promoting autophagy in human nucleus pulposus cells and thus delaying disc degeneration.

The aim of this study was to construct Chlorella mutants deficient in chlorophyll synthesis by atmospheric pressure room temperature plasma (ARTP) mutagenesis, and screen novel algal species with very low chlorophyll content which is suitable for protein production by fermentation. Firstly, the lethal rate curve of mixotrophic wild type cells was established by optimizing the mutagenesis treatment time. The mixotrophic cells in early exponential phase were treated by the condition of over 95% lethal rate, and 4 mutants with the visual change of colony color were isolated. Subsequently, the mutants were cultured in shaking flasks heterotrophically for evaluation of their protein production performance. P. ks 4 mutant showed the best performance in Basal medium containing 30 g/L glucose and 5 g/L NaNO₃. The protein content and productivity reached 39.25% dry weight and 1.15 g/(L·d), with an amino acid score of 101.34. The chlorophyll a content decreased 98.78%, whereas chlorophyll b was not detected, and 0.62 mg/g of lutein content made the algal biomass appear golden yellow. This work provides a novel germplasm, the mutant P. ks 4 with high yield and high quality, for alternative protein production by microalgal fermentation.
Chlorella/metabolism* ; Chlorophyll A/metabolism* ; Plant Breeding ; Mutagenesis ; Chlorophyll/metabolism* ; Biomass ; Microalgae