Discussion
Inflammation response of neuroimmune cells and its induced apoptotic cell death of neurons are two main pathophysiological mechanisms of secondary brain injury (SBI) after IS45,46. Previous studies had showed that many drugs, which are extracted from plants, targeting inflammatory injury and apoptotic cell death are useful therapy for IS-induced SBI47. Recently, as a safe “rare sugar” for consumption by mammals, D-allose has been developed a potential drug for cancer and different tissues ischemia/reperfusion(I/R) injury4,48-51. However, the relationships between D-allose, inflammation and apoptosis have not been elucidated. Taken together, this current study showed the following novel findings: Firstly, D-allose administration after IS was beneficial to reduce cell cytotoxicity, inflammation and neuronal apoptosis, as well as improve neurological function in vitro and in vivo. Secondly, Gal-3 was directly combined with TLR4 and activated TLR4/PI3K/AKT signaling pathway in OGD/R and MCAO/R models. Thirdly, D-allose regulates TLR4/PI3K/AKT signaling to attenuate neuroinflammation and apoptosis by inhibiting Gal-3 after IS in HT22 cells and mice.
IS often causes inflammatory injury, neuronal death and neurological deficiency. Growing evidence indicated that D-allose exerts distinct cytoprotective effects in different organ I/R injuries. However, the mechanism underlying the protective effect of D-allose in I/R injury in vitro and in vitro is still largely unclear. Previous studies indicated that D-allose reduces the production of to regulate oxidative stress in many diseases7,35,52,53. Furthermore, in our previous study, we concluded that D-allose protects brain microvascular endothelial cells from hypoxic/reoxygenated injury by inhibiting the UPR pathway and attenuating eIF2αphosphorylation and endoplasmic reticulum stress54. Moreover, similar to the previous reports about the anti-inflammation of D-allose through inhibiting LPS-induced increases in TNF-α, tissue cytokine-induced neutrophil chemoattractant-1 and myeloperoxidase concentrations, as well as the subsequent neutrophil-mediated renal, cerebral and skin slap injury. Interestingly, there are contrary results on the effect of D-allose on programmed cell apoptosis in a variety of cells that have been reported. On the one hand, D-allose induced tumor cell apoptosis in lung cancer and so on55,56. On the other, D-allose decreased apoptotic cell death in I/R-induced injury in different organs and tissues. Meanwhile, some further studies demonstrate that D-allose exerts its anti-inflammatory and anti-apoptotic effects to protect the liver57, kidney4, retina58 and skin flap51 from I/R injury. Our previous studies also provided pieces of evidence that D-allose may have therapeutic potential against cerebral I/R injury through attenuating BBB disruption, inflammatory damage and apoptosis via PPARγ-dependent regulation of NF-κB35.
Consistent with the previous reports, in the present study, we found that intraperitoneally administered D-allose markedly reduced neuron cytotoxicity, including the release of LDH concentration and cell viability in HT22 cells, diminished infarct volume ratio, brain edema, and improved neurological dysfunction in mouse after I/R injury. Additionally, we found that D-allose treatment significantly reduced cerebral I/R-induced neuronal apoptosis probably by inhibiting inflammatory response. Together, our findings suggested that D-allose had protective effects against apoptotic cell death via activating its anti-inflammation effects after I/R injury.
The next that should be noted is the mechanism of the protective effect of D-allose in vitro and in vivo after IS. Gal-3 belongs to the type three β-galectin binding lectin family and plays an important disease-exacerbating role in autoimmune/inflammatory, I/R injury and malignant diseases. Like D-allose up-, or down-regulate apoptosis in different diseases, the function of Gal-3 in apoptotic cell death is also controversial. Some reports had showed that Gal-3 contributes to the regulation of ischemic stroke by inhibiting neuronal apoptotic cell death in rats, but other studies had indicated that Gal-3 tends to modulation of glioma via enhancing tumor cell apoptosis, and increased levels of Gal-3 are found in some CNS diseases24,59-61. Meanwhile, whether Gal-3 is a proinflammatory factor or anti-inflammation protein remains unclear so far. In this study, compared with the I/R group in brains and HT22 cells, D-allose treatment significantly decreased the expression level of Gal-3, together with apoptotic markers, Bax and cleaved-Caspase3, as well as inflammatory mediators, such as cleaved Caspase1, IL-1β, IL-6 and TNF-α, suggesting reduced expression of Gal-3 induced by D-allose might mediate the neuroprotective effects we observed in this study. To confirm this, Gal-3 knockdown experiment was further performed. As the results found, both in Gal-3 down-regulate cerebral and HT22 cells, the anti-inflammation and anti-apoptosis effects of D-allose were enhanced. These results suggested that increased Gal-3 expression induced inflammatory insults and lots of neuronal apoptosis in vitro and in vivo after I/R injury, and the protective effects of D-allose following IS might be via the Gal-3 pathway.
Furthermore, one of the members of the TLRs family, TLR4, mediates several cellular processes, such as cell apoptosis, oxidative stress, infection-induced inflammation and sterile inflammation by endogenous molecules27,62. Recent shreds of evidence had found that serves as a ligand of TLR4, Gal-3 activated TLR4/NF-κB signaling to promote cell proliferation and migration in lung adenocarcinoma and reduced myocardial injury and improve cardiac dysfunction via inhibiting apoptosis63. And the other studies also revealed the TLR4-mediated pro-inflammatory and apoptotic effects of Gal-320. Additionally, PI3K/AKT signaling is an important axis regulated by ROS production and the product of metabolism, which participates in the regulation of oxidative stress, inflammatory response, and apoptosis in I/R injury64. TLR4 reportedly is an upstream regulator of the PI3K/AKT axis and upregulates p-PI3K and p-AKT levels resulting in the activation of TLR4/PI3K/AKT signaling, which promotes the proliferation and migration in the development of cardiac hypertrophy65. Recent research efforts have revealed Gal-3 is involved in the modulation of angiogenesis and apoptosis via inhibiting AKT signaling to enhance neuro-vascular protection and functional recovery after IS24. In this study, we first found Gal-3 directly combined with TLR4, and loss of Gal-3 reduced the rate of apoptosis and apoptotic proteins, Bax, cleaved Caspase3 and cleaved Caspase1, as well as pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α, which through inhibiting TLR4/PI3K/AKT signaling pathway in OGD/R and MCAO/R model. Therefore, as the results showed, compared I/R injury+Gal-3 KO in vitro and in vivo, the anti-inflammatory damage and anti-apoptotic cell death effects of D-allose were both suppressed in Gal-3 KO HT22 cells and mice cerebral which treatment with TLR4 agonist LPS. To our best knowledge, it is the first report to confirm that D-allose treatment inhibits TLR4/PI3K/AKT signaling pathway to significantly reduced cerebral I/R-induced neuroinflammation and neuronal apoptosis probably via attenuating Gal-3 in vitro and in vivo.
There are some limitations in our study. In terms of research objects, although HT22 cells have been widely used as an IS model in vitro for neuronal function, diverse results about the effects of D-allose in IS may be observed in primary neurons. In an aspect of the mechanism study, the exact mechanism of Gal-3 activating TLR4/PI3K/AKT signaling was not identified. Therefore, we need to do more studies in other neuronal cells to mimic IS model in vitro would obtain the identification of D-allose neuroprotection in I/R injury and sheds light on an accurate anti-inflammatory and anti-apoptotic mechanism.
In conclusion, our findings in mouse brains and HT22 cells suggest that D-allose attenuates post-I/R brain damage, neuronal cytotoxicity and apoptosis via reducing inflammation. This protective effect appears to be largely due to the regulated Gal-3/TLR4 complex inhibiting TLR4/PI3K/AKT signaling axis in SBI of IS (Fig. 8 Created with BioRender.com.). Therefore, D-allose may serve as a promising candidate for the treatment of IS patients.
Authors’ contributions: YL, LZ and DG designed the project and reviewed the manuscript; YL, JC, YF performed the experiments; YL, MZ, MG and XL analyzed the data; YL, MZ, YZ and JB interpreted the data; YL, LZ and DG drafted and edited the manuscript. All authors read and approved the final.
Funding: This work was supported by the National Natural Science Foundation of China (No.81971227).