Bone mass is mainly determined by proliferation and death of bone cells, one important cause of low bone mass is increased osteoblast apoptosis induced by saturated fatty acids (Zhong et al. 2011). Treatment with a long-acting LRG increased bone formation by 16% and prevented bone loss in obese women who lose weight (Iepsen et al. 2015), but the exact mechanism remains unclear. Our results showed that LRG attenuates PA-induced apoptosis via GLP1R-mediated PKA/β-catenin/Bcl-2/Bax pathway, while possibly enhances PA-inhibited differentiation by regulating the expression of OPG and RANKL (Fig. 5).
It has been reported that PA reduced cell survival and induced apoptosis in a dose- and time-dependent manner in human fetal osteoblasts hFOB1.19, the dose and time of PA incubation were 0.1, 0.25 and 0.5mmol/L for 24, 48 and 72 h (Ji-Eun et al. 2008). In the present study, we demonstrated that PA treatment ranging from 0.4 mmol/L to 1 mmol/L for 24h inhibited the proliferation, promoted ROS overproduction and apoptosis in MC3T3-L1 cells. Previously MC3T3-E1 cells were treated with 0.39–1.95 mmol/L of PA to induce apoptosis; then 0.78 mmol/L PA incubation for 12 h was chosen to study the effect of lycium barbarum polysaccharides (Jing et al. 2020), we speculated that the difference in concentration and time of PA is mainly related to the difference in cell line and the culture serum. Lee et al demonstrated that PA exerts lipotoxicity in Schwann cells by apoptosis and ROS generation, which was consistent with our results (SungWon et al. 2018).
Apoptosis of osteoblast has been studied intensively for more than 30 years, but the mechanism of PA-induced apoptosis in osteoblast has not been fully elucidated (Zhong et al. 2011; Ji-Eun et al. 2008; Jing et al. 2020; Ahmed et al. 2020-Lei et al. 2018). NF-κB, impaired activation of extracellular signal-regulated kinase (ERK), autophagy, ROS, endoplasmic reticulum (ER) stress and Fas/Jun kinase (JNK) have all been implicated in fatty acid-induced apoptosis in various settings; however, their relative role may be cell-type dependent. Caspase-3, as one of the executioner caspases, can be activated by ROS and the imbalance of pro-apoptotic protein Bax and anti-apoptotic Bcl2 in mitochondria (Peterson et al. 2008). In the present study, the protein levels of Bax, Bcl-2 and C-Capase3 were increased with the concentration of PA, which meant that PA-induced apoptosis was related to the activation of Bax/Bcl-2/Caspase3 pathway. This is the first report on the role of Bax/Bcl-2/Caspase3 in PA-induced apoptosis in MC3T3-E1 cell line.
LRG showed 97% homology with naturally active GLP-1, so it has a comparable safety profile. our results suggested that LRG at 100 and 1000 nmol/L for 72h promoted cell viability, whereas higher concentration of LRG (10000 nmol/L) showed significant toxicity to MC3T3-E1 cells. Cytotoxicity of LRG was determined by the release of lactate dehydrogenase and showed that no significant cytotoxicity in the 1-1000 nmol/L concentration range (Pang et al. 2020). Combined with the results of mRNA expression analysis of GLP1R, pretreatment with 100nmol/L LRG for 48h was selected as the optimal condition for subsequent cell experiments, which was consistent with the research results of Sun and Wu et al. (Sun et al. 2020; Wu et al. 2017).
There has been one report demonstrating LRG ameliorated the PA-induced oxidative stress, apoptosis, and endothelin-1 secretion disorder in mouse islet microvascular endothelial cells (IMECs) through GLP-1R/PKA and GTPCH1/eNOS signaling pathways (Yunyi et al. 2020). LRG pretreatment enhanced cell viability with reduced apoptosis via increasing the expression of Bcl-2 and reducing the expression of Bax in human neuroblastoma SH-SY5Y cells induced by H2O2 (Zheng et al. 2019). Our results indicated that LRG alleviated PA-induced ROS and apoptosis involved in Bax/Bcl2/Caspase3 pathway through GLP1R/PKA, which was similar as in mouse IMECs and human neuroblastoma SH-SY5Y cells respectively.
OPG and RANKL represent the factors that promote and inhibit osteoblast differentiation respectively, while their expression are regulated by β-Catenin (Bu et al. 2020). PA has been reported to inhibit differentiation, bone nodule formation and mineralization by reducing the transcriptional activities of β-catenin in human osteoblasts (Gunaratnam et al. 2014). PA also inhibited the differentiation of fetal rat calvarial cells (Yeh et al. 2014). Recently PA was reported to inhibit the proliferation, ALP activity, production of procollagen type I C-peptide and mineralization in the human periodontal ligament stem cells (Takeuchi et al. 2020). In the present study, 0.4 mmol/L PA reduced the OPG expression and phosphorylation of β-catenin (Ser675), meanwhile promoted RANKL expression, which was consistent with the previous reports.
Multiple studies have shown that LRG promoted osteogenic differentiation through a signaling network of Notch/Wnt/Hedgehog signaling pathways (Hou et al. 2020), or by ERK5 pathway (Sun et al. 2020), or via regulating the expression of Smad2/3 through PI3K/Akt and Wnt/β-Catenin pathways in MC3T3-E1 cells (Liu et al. 2018). In the present study, we found that LRG-mediated activation of β-catenin resulted in transcriptional activation of OPG and transcriptional inhibition of RANKL, while increased cAMP levels and activation of PKA, suggesting that LRG promoted the dialogue between PKA and β-Catenin pathways in PA-treated MC3T3-E1 cells.
Continuous activation of β-Catenin by phosphorylation promotes osteocyte growth and increase bone mass in vivo and in vitro (Kramer et al. 2010). Activated PKA by cAMP phosphorylates β-catenin at Ser675 position, promoting its stability and transcriptional activity, and transferred to the nucleus (Mei et al. 2010). The results of this study showed that PKA inhibitor H89 partially attenuated phosphorylation of β-Catenin at Ser675 by LRG, thus promoted activation of Bax/Bcl2/Caspase3 and apoptosis of MC3T3-E1 cells possibly via inhibiting mitochondrial ROS production. ROS is mainly produced in mitochondria, so elevated intracellular ROS levels mean elevated ROS in mitochondria. β-catenin knockout in MC3T3-E1 cells is required to further confirm the central role of β-catenin in LRG' lipotoxic protective effect.
Effect of LRG is related with GLP1R under most circumstances (Xu et al. 2021; Liu et al. 2018). Our results proved that LRG promoted GLP1R expression, accompanied with activation of cAMP-PKA/β-catenin pathway, while Exendin9-39 partially antagonized the effects of LRG on cell proliferation, anti-apoptosis, ROS reduction and activation of cAMP-PKA/β-catenin pathway in PA-treated MC3T3-E1 cells, which implied that at least part of the protective effect of LRG was achieved through GLP1R activation. GLP1R knockdown via siRNA will be performed in follow-up experiments to confirm the mechanism of LRG action in PA-treated MC3T3-E1 cells. Also the results of present study need to be repeated in animal model. All these limitations should be addressed in future studies.
The present study confirmed that LRG promotes proliferation of MC3T3-E1 cells and inhibits the PA-induced ROS production and apoptosis through PKA/β-Catenin/Bcl-2/Bax signaling pathway that includes GLP1R activation, accompanied by improvement of bone cell function. Our results point to LRG as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions.