To investigate the protective effects of curcumin on DM/CCH-induced pathologies, our previous studies have successfully established stable models in vivo and in vitro[11, 38]. Specifically, we subjected rats to a HFD and low-dose STZ combined with BCCAO to model DM/CCH in vivo, whereas we established a corresponding model in vitro by subjecting BV2 microglia to hypoxia and a high-glucose environment. The present study revealed that curcumin treatment effectively protected against DM/CCH-induced cognitive dysfunction, as well as attenuated neuronal injury and death in the CA1, CA3, and DG regions of the hippocampus. Molecular-biology analysis revealed that the underlying mechanisms of curcumin’s protective effects were associated with inhibiting neuroinflammation, regulating the TREM2/TLR4/NF-κB pathway, suppressing neuronal apoptosis, and mitigating NLRP3-dependent pyroptosis.
CCH plays a pivotal role in the progression of cognitive impairments in VaD, as it can induce neuroinflammation, decrease energy supplementation, contribute to neuronal injury, and impair memory[18, 49, 50]. Importantly, a previous study has demonstrated that diabetes aggravates neuronal apoptosis and accelerates cognitive dysfunction. In our present study, in the MWM, the DM/CCH group exhibited impairments in spatial learning and memory, as revealed by a prolonged escape latency, decreased number of crossings over the original platform location, and less time spent in the target quadrant; these findings are consistent with those of previous reports[11, 52, 53]. However, long-term curcumin treatment significantly ameliorated cognitive dysfunction. Moreover, we found that curcumin attenuated DM/CCH-induced neuronal damage, as there was an increase in NeuN-positive cells and HE staining showed a decrease in atrophied neurons and dark-stained nuclei; these results were consistent with our behavioral results in the MWM. Collectively, these findings indicate that curcumin protected against cognitive impairments and attenuated neuronal damage in DM/CCH rats.
Accumulating evidence has demonstrated that CCH induces significant inflammatory responses, including glial activation and excessive production of pro-inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α), which are pathological signatures that are closely related to neuronal damage. Moreover, previous studies have also found that DM increases microglial activation and the release of pro-inflammatory cytokines[55, 56]. Taken together, these findings suggest that neuroinflammation might play a vital role in the occurrence and development of DM combined with CCH. Consistent with these previous results, in our present study, we found overactivation of microglia and excessive production of pro-inflammatory cytokines in DM/CCH rats. However, curcumin treatment dramatically inhibited neuroinflammation, as indicated by decreased Iba1-positive cells and decreased production of pro-inflammatory cytokines compared to those in DM/CCH rats. Our results in vitro also indicated that curcumin reversed high-glucose/hypoxia-induced increases in pro-inflammatory cytokines. CCH causes a cascade of pathological processes in which many different signaling pathways are activated, including the TREM2/TLR4/NF-κB pathway. TLR4, a classic pattern-recognition receptor, is mainly expressed in microglia and is stimulated by its cognate ligands. Following stimulation, TLR4 further activates the NF-κB pathway, which plays an important role in the transcription of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Recently, several studies have demonstrated that TREM2 downregulates TLR4 signaling[19, 58, 59]. As a membrane-bound receptor mainly expressed in microglia of the central nervous system, TREM2 has been confirmed to regulate the primary functions of microglia, such as inhibiting pro-inflammatory cytokines, promoting phagocytosis, and maintaining the energy metabolism of microglia[60–62]. A recent study found that lack of TREM2 amplified TLR4-driven inflammatory responses. Moreover, in-vivo studies have demonstrated that TREM2 attenuates neuroinflammation by negatively regulating TLR4-mediated activation of NF-κB signaling. In our present study, we found that DM/CCH induced activation of the TLR4/NF-κB pathway, and unexpectedly also increased the expression of TREM2. This latter finding is contrary to that of some previous studies[20, 64], perhaps due to different models or different observation times. Interestingly, in our present study, curcumin treatment effectively reversed DM/CCH-induced increase in TLR4 and NF-κB levels while further increasing TREM2 expression. Our results in vitro further confirmed these findings. Recently, Takalo et al discovered that phospholipase C gamma 2 (PLCγ2) may be associated with TREM2/TLR4 signaling. They found that PLCγ2 was activated downstream of TREM2 to promote beneficial microglial function, while PLCγ2 also acted downstream of the ligand-stimulated TLR pathway to induce inflammatory responses. In the absence of TREM2, microglia are unable to play a protective role in the TREM2/PLCγ2 pathway and eventually amplify activation of downstream signaling pathways of TLR. However, whether PLCγ2 is associated with curcumin-mediated regulation of TREM2/TLR4 signaling requires further investigation.
Apoptosis plays a major role in neurological diseases, including AD, PD, and VaD[22, 66, 67]. Many signaling pathways regulate apoptosis, including Notch, Bax/Bcl2/caspase3, and IRE1-α/TRAF2/ASK1 pathways[68–70]. Among them, Bax/Bcl2/caspase3 signaling has been extensively studied. Bax is a pro-apoptotic Bcl2-family protein, while Bcl2 is an anti-apoptotic protein. Additionally, activation of caspase3 is a key event in the execution of DNA fragmentation factor (DFF). The activation of DFF then activates endonucleases, cleaves nuclear DNA, and ultimately leads to cell death. Dysregulation of apoptotic pathways can cause cell death and promote disease progression. A recent study found that Bax protein expression was increased while Bcl2 expression was decreased in a mouse model of PD. Consistently, our present results showed that expression levels of cleaved-caspase3 and Bax were significantly increased whereas Bcl2 levels were significantly decreased following DM/CCH in vivo, whereas these changes in expression levels were reversed by curcumin treatment. Similar results were observed in high-glucose/hypoxia-treated microglia in vitro. Therefore, our findings suggest that curcumin alleviated DM/CCH-induced and high-glucose/hypoxia-induced pathologies through reducing apoptosis via the Bax/Bcl2/caspase3 pathway.
Recently, inflammasomes have been reported to be associated with the progression of neurodegenerative disorders[47, 73–75]. Inflammasomes are large polyprotein complexes that mediate the innate immune response to infectious microorganisms. At present, researchers have found a variety of inflammasomes, such as NLRP1, NLRP3, NLRP6, NLRP7, NLRC4, and AIM2. Among them, the NLRP3 inflammasome is the best characterized and includes a sensor (NLRP3), an adaptor protein (ASC), and an effector protein (caspase1)[77, 78]. After activation, the NLRP3 inflammasome contributes to the conversion of pro-caspase1 into active caspase1, resulting in the production of proinflammatory cytokines (e.g., IL-18 and IL-1β), thereby activating NLRP3 signaling that aggravates neuroinflammation and can promote the progression of neurodegenerative diseases[79, 80]. Additionally, active caspase1 can induce pyroptosis via cleavage of GSDMD. As a protein substrate of caspase1, GSDMD has been identified as a key substrate of the inflammasome proteolytic pathway and the main driver of NLRP3-dependent pyroptosis. After cleavage of GSDMD, there is an N-terminal cleavage product that remains, which is sufficient to form membranous cores. Therefore, pyroptosis is distinct from apoptosis since it is characterized by membrane rupture that causes the release of cytosolic contents to further aggravate inflammatory responses. A recent study has found that NLRP3/caspase1/GSDMD signaling is overactivated in PD. In our present study, we detected the expression of NLRP3-signaling-associated proteins in vivo and in vitro. Our results in vivo showed that curcumin effectively suppressed DM/CCH-induced overexpression of NLRP3, ASC, cleaved-caspase1, IL-18, and cleavage of GSDMD. Similar results were found in microglia exposed to high glucose combined with hypoxia in vitro. Taken together, these findings suggest that the beneficial effects of curcumin involve modulation on NLRP3-induced pyroptosis. However, the specific mechanism of curcumin suppressing pyroptosis requires further investigation.