Cerebral ischemia/reperfusion (CIRI) is a leading cause of death worldwide. A small GTPase known as ADP-ribosylation factor-like protein 13B (ARL13B) is essential in several illnesses. The role of ARL13B in CIRI remains unknown, though. A middle cerebral artery occlusion/reperfusion (MCAO/R) in rats as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) models in PC12 cells were constructed. The neuroprotective effects of ARL13B against MCAO/R were evaluated using neurological scores, TTC staining, rotarod testing, H&E staining, and Nissl staining. To detect the expression of proteins associated with the SHH pathway and apoptosis, western blotting and immunofluorescence were employed. Apoptosis was detected using TUNEL assays and flow cytometry. There was increased expression of ARL13B in cerebral ischemia/reperfusion models. However, ARL13B knockdown aggravated CIRI nerve injury by inhibiting the sonic hedgehog (SHH) pathway. In addition, the use of SHH pathway agonist (SAG) can increased ARL13B expression, reverse the effects of ARL13B knockdown exacerbating CIRI nerve injury. ARL13B alleviated cerebral infarction and pathological injury and played a protective role against MCAO/R. Furthermore, ARL13B significantly increased the expression of SHH pathway-related proteins and the anti-apoptotic protein BCL-2, while decreased the expression of pro-apoptotic protein BAX, thus reducing apoptosis. The results from the OGD/R model in PC12 cells were consistent with those obtained in vivo. Surprisingly, we demonstrated that ARL13B regulates the cell cycle to protect against CIRI nerve injury. Our findings indicate that ARL13B protects against CIRI by reducing apoptosis through SHH-dependent pathway activation, and suggest that ARL13B plays a crucial role in CIRI pathogenesis.

We have previously demonstrated a rapid secretion of matrix metalloproteinase-2 (MMP-2) in the ischemic brain. Since Scube2 can interact with Sonic hedgehog (Shh) to maintain blood-brain barrier (BBB) integrity via regulating the interaction between brain capillary endothelial cells (ECs) and perivascular astrocytes, and it is also a substrate of MMP-2, we hypothesized that the secreted MMP-2 could degrade Scube2 and contribute to ischemic BBB disruption. Using an in vitro ischemic model of 90-min oxygen-glucose deprivation/3-h reoxygenation (OGD/R) and an in vivo mouse stroke model of 90-min middle cerebral artery occlusion (MCAO) with 3-h reperfusion, we established an important role of MMP-2-mediated Scube2 degradation in early ischemic BBB disruption. Exposure of C8-D1A cells and bEnd.3 cells to OGD/R increased MMP secretion in both cells, and C8-D1A cells appeared to secrete more MMPs than bEnd.3 cells. Co-IP and double-immunostaining revealed that Scube2 co-localized well with MMP-2 in C8-D1A cells and could be pulled down by MMP-2 antibodies. In MCAO mice, Scube2 protein showed a drastic reduction in ischemic brain tissue, which was accompanied by suppressed expression of Shh and its downstream molecules. Of note, specific knockdown of astrocytic Scube2 with AAV-shScube2 augmented MCAO-induced Shh suppression and exacerbated BBB leakage and inflammatory reactions in the ischemic brain. Last, incubation of bEnd.3 cells with conditioned medium derived from OGD-treated C8-D1A cells led to a significant inhibition of the Shh pathway in bEnd.3 cells and degradation of VE-cadherin and ZO-1. Inhibition of MMP-2 with SB-3CT or over-expression of Scube2 with plasmids in C8-D1A cells alleviated the above effect of C8-D1A cells-derived conditioned medium. Taken together, our data indicate that ischemia-induced secretion of MMP-2 may contribute to early BBB disruption in ischemic stroke via interrupting the shared Scube2-Shh pathway between brain capillary ECs and perivascular astrocytes.

Disruption of intervertebral disc (IVD) homeostasis caused by oxidative stress and nucleus pulposus cell (NPC) senescence is a main cause of intervertebral disc degeneration (IDD). The sonic hedgehog (Shh) pathway plays an important role in IVD development, but its roles in IDD are unknown. This study aimed to investigate the effects of the Shh pathway on the alleviation of IDD and the related mechanisms. In vivo, the effect of the Shh pathway on IVD homeostasis was studied by intraperitoneal injection of recombinant Shh (rShh) and GANT61 based on puncture-induced IDD. GANT61, lentivirus-coated sh-Gli1 and rShh were used to investigate the role and mechanism of the Shh pathway in NPCs based on senescence induced by Braco19 and oxidative stress induced by TBHP. Shh pathway expression decreased, and senescence and oxidative stress increased with age. Intraperitoneal injection of rShh activated the Shh pathway to suppress oxidative stress and NPC senescence and consequently alleviated needle puncture-induced IDD. In vitro, the Shh pathway upregulated glutathione peroxidase 4 (GPX4) expression to suppress oxidative stress and senescence in NPCs. Moreover, GPX4 suppression in NPCs by si-GPX4 significantly reduced the protective effect of the Shh pathway on oxidative stress and senescence in NPCs. Our results demonstrate for the first time that the Shh pathway plays a key role in the alleviation of IDD by suppressing oxidative stress and cell senescence in NP tissues. This study provides a new potential target for the prevention and reversal of IDD.

Adamantinomatous craniopharyngioma (ACP) is a neuroendocrine tumor whose pathogenesis remains unclear. This study investigated the role of glioma-associated oncogene family zinc finger 1 (GLI1), a transcription factor in the sonic hedgehog (SHH) signaling pathway, in ACP. We discovered that GLI1 regulates the expression of IL-6, thereby triggering inflammatory responses in ACP and influencing the tumor\'s progression. Analyzing the Gene Expression Omnibus (GEO) database chip GSE68015, we found that GLI1 is overexpressed in ACP, correlating positively with the spite of ACP and inflammation markers. Knockdown of GLI1 significantly inhibited the levels of tumor necrosis factor α, interleukin-6 (IL-6), and IL-1β in ACP cells, as well as cell proliferation and migration. We further identified a binding site between GLI1 and the promoter region of IL-6, demonstrating that GLI1 can enhance the expression of IL-6. These findings were verified in vivo, where activation of the SHH pathway significantly promoted GLI1 and IL-6 expressions in nude mice, inducing inflammation and tumor growth. Conversely, GLI1 knockdown markedly suppressed these processes. Our study uncovers a potential molecular mechanism for the occurrence of inflammatory responses and tumor progression in ACP.

Gastric cancer is one of the deadliest malignant tumors, and half of the patients develop recurrences or metastasis within 5 years after eradication therapy. Cancer stem cells (CSCs) are considered to be important in this progress. The sonic hedgehog (SHH) pathway plays an important role in the maintenance of gastric CSCs characteristics. The p63 proteins are vital transcription factors belonging to the p53 family, while their functions in regulating CSCs remain unclear. The preventive effects of dietary diallyl trisulfide (DATS) against human gastric cancer have been verified. However, whether DATS can target gastric CSCs are poorly understood. Here, we investigated the role of ΔNp63/SHH pathway in gastric CSCs and the inhibitory effect of DATS on gastric CSCs via ΔNp63/SHH pathway. We found that ΔNp63 was upregulated in serum-free medium cultured gastric tumorspheres compared with the parental cells. Overexpression of ΔNp63 elevated the self-renewal capacity and CSC markers\' levels in gastric sphere-forming cells. Furthermore, we found that ΔNp63 directly bound to the promoter region of Gli1, the key transcriptional factor of SHH pathway, to enhance its expression and to activate SHH pathway. In addition, it was revealed that DATS effectively inhibited gastric CSC properties both in vitro and in vivo settings. Activation of SHH pathway attenuated the suppressive effects of DATS on the stemness of gastric cancer. Moreover, DATS suppression of gastric CSC properties was also diminished by ΔNp63 upregulation through SHH pathway activation. These findings illustrated the role of ΔNp63/SHH pathway in DATS inhibition of gastric cancer stemness. Taken together, the present study suggested for the first time that DATS inhibited gastric CSCs properties by ΔNp63/SHH pathway.

BACKGROUND: The omnipresence of human phthalate (PAE) exposure is linked to various adverse health issues, including breast cancer. However, the effects of low-dose PAE exposure on breast cancer stem cells (BCSCs) and the underlying mechanism remain unexplored.
METHODS: BCSCs from breast cancer cell lines (MDA-MB-231 and MCF-7) were enriched using a tumorsphere formation assay. Gene and protein expression was detected by measurement of quantitative real-time reverse transcription PCR, western blot, and immunofluorescence assays. Transient transfection assays were used to evaluate the involvement of Gli1, a signaling pathway molecule and ΔNp63α, an oncogene in influencing the PAE-induced characteristics of BCSCs.
RESULTS: PAE (butylbenzyl phthalate, BBP; di-butyl phthalate, DBP; di-2-ethylhexyl phthalate, DEHP) exposure of 10-9 M significantly promoted the tumorsphere formation ability in BCSCs. Breast cancer spheroids with a 10-9 M PAE exposure had higher levels of BCSC marker mRNA and protein expression, activated sonic hedgehog (SHH) pathway, and increased mRNA and protein levels of an oncogene, ΔNp63α. Furthermore, suppression of the SHH pathway attenuated the effects of PAEs on BCSCs. And the overexpression of ΔNp63α enhanced PAE-induced characteristics of BCSCs, while low expression of ΔNp63α inhibited the promotion effects of PAEs on BCSCs and the SHH pathway.
CONCLUSIONS: Low-dose PAE exposure promoted the stem cell properties of BCSCs in a ΔNp63α- and SHH-dependent manner. The influence of low-dose exposure of PAEs and its relevance for the lowest observed effect concentrations requires further investigation, and the precise underlying mechanism needs to be further explored.