Amyloid beta (Aβ) plaque consists of aggregative and neurotoxic peptides generated from amyloid precursor protein (APP) contribute to the pathological progression of neurodegeneration lead to Alzheimer’s disease [4], involves in generation of free radicals, activation of proinflammatory genes, oxidation of macromolecules, organelle dysfunction and apoptosis of neurons. Behind the onset of amyloid plaques formation, oxidative stress imparts role that induce brain cholesterol oxidises into oxysterols [59, 60]. 7-ketocholesterol, one of the predominant oxysterols, when accumulates in the neuronal cells which cleaves the amyloid precursor protein into neurotoxic amyloid fragments that leads to pathological process of Alzheimer’s disease [13].
7KCh in this study was employed to induce amyloidogenic initiation and the consequent disease-progressing events in AD. Bornyl acetate and menthol monoterpenes were reported to be an antioxidant and anti-inflammatory potential in various studies [25, 32, 61]. In the present study, we investigated the anti-amyloidogenic and neuroprotective role of bornyl acetate and menthol against 7KCh-induced SH-SY5Y neuroblastoma cells.
7KCh molecules demonstrated their potential cytotoxic activity, disrupting cellular homeostasis and inducing death in PC12 cells [62], hCMEC/D3 human brain endothelial cells [63]. 7KCh induced cytotoxic effect in BV-2 microglial through mitochondrial dysfunction [64]. Similarly, in our results the cytotoxic effect of 7-ketocholesterol-induced in SH-SY5Y neuronal cells were also observed. The effect of bornyl acetate and menthol tested on SH-SY5Y cells exhibited no significant toxicity. Consistently, the earlier reports of bornyl acetate from essential oils showed neuroprotective effects in okadaic-acid induced PC12 cells [29]. Similarly, menthol have been shown effective cell survival at 0.2 µM concentration in SH-SY5Y neuronal cells against cisplatin and Aβ1–42 induced cytotoxicity [39]. Supporting the previous evidence, bornyl acetate and menthol co-treated cells inhibited the cellular loss induced upon 7KCh. Prior studies investigated on compounds from essential oils α-pinene, 1,8-cineole attenuated the loss of PC-12 cellular viability through redox balance on cells [65, 66]. Paeoniflorin, provided protection against amyloid-induced cytotoxicity in SH-SY5Y cells [67].
External stimuli like pathogens, toxins and injury cause an immune response called inflammation which is one of the events that initiate the healing mechanism, that in prolonged conditions causing free radical generation, cellular membrane damage and protein denaturation. RBCs under the condition of excess fluid accumulation and membrane damage, are more prone to secondary damage in case of inflammation. 7KCh molecules, due to their integration into the lipid bilayers have cytotoxic characteristics [68]. In this investigation, the compounds were tested for their anti-inflammatory efficacy by their effect on RBC membrane stabilization ability. Human RBC haemolysis induced in the hypotonic solution, which was suppressed by compounds bornyl acetate and menthol exerting a protective role on the cellular membrane integrity. Many natural extracts and derived components are elucidated for their anti-inflammatory activity in vitro by hypotonicity-induced haemolysis in human RBCs [42]. Essential oils are established for their activity of stabilized the RBC membrane integrity as well as haemoglobin release [69] especially, monoterpenes from essential oils such as, terpinene-4-ol, eucalyptol exhibited their anti-inflammatory activity of human RBC stabilization [70, 71, 72].
Oxysterol accumulation in systemic circulation and brain act as the immediate precursor neurodegenerative diseases [73]. Especially 7KCh accumulation in cells could contribute to cellular stress, organelle dysfunction, inflammation and cell death [74]. 7KCh exposure favours lipid accumulation with an exacerbated level of neutral and polar lipids in U937 cells that evokes cell death through various signal transduction [75] and the inhibitory activity was analysed by monoterpenes sarcoglarols, lindenane heterodimers and citral that reduced intracellular accumulation of lipid content in the L02 cells exposed to free fatty acids as well as 3T3-L1 cells, respectively [76, 77]. Oil red O, a lipophilic dye that binds to neutral lipids in living things, used for the assessment of lipid droplets in BV-2 microglial cells [78]. Likewise, in the present study examined for lipid accumulation and our results revealed that 7KCh treated cells shows the greatest lipid accumulation. The cells treated with bornyl acetate or menthol reduced lipid uptake by cells which indicates their potent property of inhibiting lipid accumulation. Our results coincided with previous results of bornyl acetate incited inhibitory effect against the accumulation of OxLDL molecule that induces attachment of monocytes in the human umbilical endothelial cells [27]. In addition, the ability of preventing lipid accumulation on neuronal cells could possibly alleviate the neurodegenerative pathology.
Reactive nitrogen species (RNS) and reactive oxygen species (ROS) are free radicals that can generate toxic by-products and activate inflammatory pathways and organelle malfunction causing neuronal loss [79]. Such prooxidant radicals act as mediators of oxidative stress that possess many physiological effects and increase in oxygen radical generation causing Aβ peptides accumulation which has been reported in pathologies [80]. Phospholipids, proteins and nucleic acids undergo oxidative damage through the generation of free radicals. According to many studies oxidative stress induced by free radicals enhances the production of β-secretase and causes the accumulation of amyloid plaque and neurodegeneration through various signalling pathways [81]. 7KCh increased free radical generation in PC12 cells reported in the previous study [61]. Our experimental reliably showed 7KCh induced generation of nitric oxide and reactive oxygen species which caused cellular alterations. There are versatile natural compounds have been reported previously, that could scavenge the free radicals of cellular metabolism, reduced the ROS generation in neuronal cells [65, 82]. Prior findings also stated that bornyl acetate and menthol have anti-inflammatory and antioxidants properties [61]. Supporting those evidence, in our study monoterpenes bornyl acetate or menthol treated groups exhibited a decrease in nitric oxide and ROS generation.
Lipid accumulation and free radical generation disturb the neurotransmitter balance. A decline in acetyl choline (Ach) level is associated with cognitive impairment and mental deterioration which is implicated in the case of Alzheimer’s disease. This decline in Ach level is caused due to the activity of catabolic enzyme, acetylcholinesterase. Hence, numerous inhibitors of these enzymes are currently available as symptomatic treatment of onset and progression of Alzheimer’s disease [83]. There are natural cholinesterase inhibitors existing as phytochemicals and other biological sources like monoterpenes, sesquiterpenes attributed to anti-acetylcholinesterase activity [84]. Among which some of them are α-pinene, terpineol, linalool, ß-myrcene, nerol and geraniol which strongly inhibited acetylcholinesterase enzyme in both in vitro and in vivo condition [85]. Hypothesis raised on the cholinesterase inhibitors to counteract amyloid beta and tau accumulation. In this context, the cholinesterase activity was analysed against 7KCh induced SH-SY5Y cells, expressed the highest level of acetylcholinesterase activity that could contribute to plaque generation. However, our results claim the anti-cholinergic efficacy of bornyl acetate and menthol which suppressed the elevated levels of AChE induced by 7KCh exposure in SH-SY5Y cells, thus proving their anticholinesterase efficacy against 7KCh.
Aggregation and accumulation of insoluble proteinaceous amyloid peptides into amyloid plaque is the primary neuropathological hallmark of Alzheimer's disease. Amyloid deposits are caused by aggregations of the amyloid-peptide [86]. Amyloid plaque is formed due to amyloid precursor protein interrupted by deposits of altered lipid structures like oxysterols in the cell membrane which cleaves APP into insoluble aggregative amyloid peptides. The Aβ42 aggregates in the extra cellular spaces that prevent synaptic transmission from one cell to other cells and neuronal cells [87]. Our investigation deals with the peptide oligomers and the presence of amyloid fibrils or aggregates in the 7KCh induced cells. Contrastingly, the cells co-treated with bornyl acetate and menthol decreased fluorescence due to poor thioflavin t bonding and amyloid formation. From the result it is evident that both compounds possess potential anti-amyloidogenic properties. Similarly, the previous reports with monoterpenes of various origin claim to have the anti-amyloidogenic property in in vitro and in vivo models [88]. Supporting our results, monoterpenes alpha-terpeniol, myrcene checked against Aβ fibril formation, counteracted AD consequences by reducing fibril formation [89, 90].
The endoplasmic reticulum (ER) takes over the charges of protein folding mechanism and intracellular signalling like calcium homeostasis [91]. Calcium ion (Ca2+) is finely controlled by neurons to relay information. Ca2+ homeostasis is essential for the survival and performance of neurons [92]. 7KCh are the vigorous inducer of cellular as well as ER stress through protein kinases by the immediate activation of TLR4 receptor which involves alteration in the intracellular calcium influx [15]. Stress response by 7KCh induced the influx of intracellular calcium levels in PC12 cells leading to necrosis or apoptosis [61]. 7KCh treatment in neuronal cells inflicted the intracellular calcium deposition with upregulated ATF-6, HSP70 (GRP78) genes as an indication of ER stress and protein misfolding which was comparable to prior study results examined in neuronal cells and these deleterious effects was ameliorated by kimchi extracts [93]. Terpene glycoside from root bark of Paeonia suffruticosa treated endoplasmic related inflammatory responses where the ER-stress related factors GRP78/ATF-6, XBP-1 are downregulated [94]. Monoterpenes thymoquinone protected liver injury by lowering ER stress indices GRP78, CHOP and caspase-12 expression which may be related to its antioxidant qualities as well as the attenuation of oxidative stress, mitochondrial damage and apoptosis. In addition, thymoquinone reduced the release of cytochrome C and the activity of caspase apoptotic markers [95]. The effect of terpinene-4-ol was also reported for amelioration of ER stress and activating factors [96]. Numerous papers described how natural substances can affect the signalling between ER and calcium homeostasis [97]. Evidently, the compounds bornyl acetate and menthol under our investigation, showed calcium reduction in 7KCh-induced cells associated with the decrease in gene expression of HSP70/ATF-6.
Mitochondria regulates the processes like oxygen consumption, ATP synthesis, oxygen radical generation, calcium mobilization and neuronal viability. The dysfunctional state of mitochondria alters the redox balance, disrupts electron transport, generates reactive oxygen species, initiates oxidative phosphorylation and releases caspase-activating proteins [98]. Several studies reported the alteration of mitochondrial membrane stability and integrity in N2a, PC12 cells by 7KCh molecule that mediates cell death due to an increase in ROS and calcium levels [61, 99]. In the same manner, our results showed that 7KCh-induced mitochondrial dysfunction due to the depolarization of mitochondrial membrane potential in SH-SY5Y cells. Constantly, the results from our study suggest that bornyl acetate and menthol co-treatment showed the mitochondria membrane potency indicating the protection against 7KCh-induced mitochondrial damage. Previous study reported the stability of mitochondrial membrane potential retained by a monoterpene phenol compound named thymol in lung fibroblast cells V79 [100], paeoniflorin that suppressed mitochondria mediated apoptosis in PC12 cells [101] and linalool provided neuroprotection by preserving the mitochondrial membrane potential in case of NMDA-induced HT-22 cells [102].
Amyloid plaque accumulation on neuronal cells experiences considerable evidence of apoptosis leading to the loss of neurons through an intrinsic pathway of mitochondrial and endoplasmic reticulum malfunction leads to AD progression also execute neurodegeneration through apoptosis [103]. 7KCh-induced oxidative stress augmented DNA damage forming 8-oxoguanine, internucleosomal DNA fragmentation in RAW 264.7 macrophages ([104]. According to the previous approaches, the early apoptotic cells and necrotic cells can be distinguished by using the dual stains acridine orange and EtBr. Acridine orange is a cell-permeable fluorescent nucleic acid dye that binds to live cells and exhibits green fluorescence and ethidium bromide attaches to the base pairs of DNA’s that has lost its membrane integrity [105]. Our result showed poor membrane integrity of the 7KCh-induced apoptotic cell. Contrarily, the co-treatment of bornyl acetate and menthol reduced the apoptotic effect of 7KCh-induced neuronal cells exhibiting live cells. Biomolecules are constantly used as an anti-apoptotic agent with that the protective effect of thymol, alpha pinene, carvacrol, linalool, eucalyptol and myrcene has been established in mammalian cells [106, 107, 108]. Conspicuously, comparable results obtained in our investigation, the cellular and nuclear membrane integrity was protected by bornyl acetate and menthol.
Oxidative stress has a profound effect in the facet of many neurodegenerative diseases. Reactive oxygen radicals and other biological radicals generated in the cells are neutralized by the glutathione enzyme through a concerted cascade of enzymes glutathione peroxidases, glutathione-s transferases and glutathione reductase also regulates apoptosis, gene expression, and other signal transduction process [109]. Both peripheral tissues and the central nervous system of AD patients exhibit buildup of free radical and changes levels and activity of antioxidant enzymes [110]. Multiple evidence suggests that 7KCh-induced apoptosis in cells seems to affect the levels of glutathione enzyme [111]. In relevance to the previous reports our results comparably exhibited low anti-oxidants levels in cells exposed to 7KCh treatment. However, we found that the free radical scavenging property of bornyl acetate and menthol enhanced higher antioxidants level, demonstrating the cellular protection.
Anti-oxidant imbalance elevated by oxidative stress markers in the brain and cerebrospinal fluid in Alzheimer's disease include the lipid peroxidation by-products 4-hydroxynonenal (HNE), malondialdehyde (MDA), F2-isoprostanes and protein oxidation derivatives such as protein carbonyls and 3-nitrotyrosine [110]. 7KCh in smooth muscles produced lipid peroxides [112]. Supporting the previous study, 7KCh-induced apoptosis that contributes to lipid and protein oxidation displayed higher levels of malondialdehyde and carbonylated protein content in the cells which indicates the membrane lipid damage and protein oxidation. Noticeably, there was a considered decrease in the level of lipid and protein oxidation which was regulated by bornyl acetate and menthol treatment. On the other hand, natural sources of antioxidants are used to enhance the normal mechanism, hence the bio-active agents with one such effective derivative monoterpene has been reported to scavenge the free radical thus reducing the oxidation of other cellular molecules [113, 114]. Citronellol enhanced anti-oxidant enzymes against rotenone induced neuronal damage reduced the level of lipid peroxidation [115], reduction in advanced glycation end products were reported by 1,8-cineole [116] and dihydroactinidiolide significantly reduced lipid peroxidation and protein carbonylation in SH-SY5Y cells induced by sodium dithionite, glutamate, amyloid β and colchicine [117]. In contrast to 7KCh treatment, bornyl acetate and menthol counteracted the pro-oxidant by promoting anti-oxidant levels.
7KCh implication in the neurodegenerative diseases mediated pathological progression with persistent inflammation, induces neuronal loss through several cascade. In relation to this the gene expression of various signal transduction mediators involved in neuroinflammatory process and the efficacy of bornyl acetate and menthol against these mediators were studied. Toll like receptor 4 (TLR4) respond to the lipopolysaccharide molecules and other stimuli like 7KCh. [15]. TLR4 has grown into a possible connection underlying dyslipidemia and increased inflammatory cytokine production. Inhibition of this receptor suggested as a target for many inflammatory diseases [118]. This further supports our work that 7KCh molecules upregulation of TLR4 in the neuronal cells activates various inflammatory signals contributing to neurodegeneration. In such condition, the expression of TLR4 was downregulated upon bornyl acetate and menthol co-treatment which could suppress the activation of inflammatory progression. In this connection, previously reported phytoconstituents like lycopene, carvacrol and linalool exhibited their anti-inflammatory effect through TLR4 suppression under in vitro and in vivo conditions [119, 120, 121].
PARP-1 involves in DNA repair and regulation of astrocytes as well as microglial function. Progressive DNA damage can activate immune response through production of nitric oxide and other inflammatory cytokines involving PARP-1 cleavage in various pathological process of cell death mediated by caspases and cathepsins. These cleaved PARP fragment inhibits the DNA repair enzymes resulting in apoptosis. PARP-1 remains a powerful activator of mitochondrial dysfunction/caspase-dependent cell death via proapoptotic factors like cytochrome c and cathepsins release [122, 123]. In addition, PARP-1 overactivation was the proposed cause of neuroinflammation and apoptosis in amyloid peptide 1–42 induced condition [124]. Previous research specified that 7KCh instigated PARP-1 activity and proinflammatory conditions associated with a significant increase in pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α and COX-2. in human brain endothelial cells [125, 126] also contributed to the cell caspase dependent apoptosis in many vascular diseases [127]. Similarly, our investigation revealed the upregulation of pro-inflammatory such as IL-6, iNOS and apoptotic markers PARP-1, cathepsin D and caspase-3 by 7KCh. Interestingly, bornyl acetate and menthol co-treated cells showed reduced levels of 7KCh-induced inflammatory markers. Moreover, previous literatures pertaining to our study stated that borneol and menthol potentially reduced the expression of pro-inflammatory mediators iNOS, TNF-α and IL-1β and the major transcription factor NF-κB p65 nuclear translocation in neuronal injury and also thymol, carvacrol, linalool and linalyl acetate α-terpinene, α-terpineol, terpinen-4-ol, α-carveol, menthone, pulegone, geraniol, citral, citronellol that were counteracted many chronic inflammatory conditions [23]. Nicotinamide reduced the PARP-1 activity in amyloid beta induced Alzheimer’s condition in rat model [124].
Amyloid pathologies are regulated other way through cholesterol efflux is executed by the transporters, ABCA1 and ABCG1 that prevents the accumulation of oxysterol and its toxicity, more importantly, ABCA1 facilitates anti-aggregation of Aβ by enabling ApoE binding and neutralization of Aβ1–42 oligomer to eliminate from the brain [7, 128]. Smoke exposure reduced ABCA1 expression, impairs cholesterol efflux from macrophages in vivo and in vitro, which causes lipid-accumulation in macrophages resulting in exacerbation of pulmonary inflammation correlated with up-regulation of the TLR4 pathway [129]. In relation to this 7-ketocholesterol accumulated cells express low ABCA1 due to impaired cholesterol efflux, which was obviously increased by bornyl acetate and menthol to enable cholesterol efflux from the cell avoiding the cytotoxic effects. The natural extract of saffron containing crocins, crocetin, safranal enhanced the activity of ApoE and ABCA1 transporters thus regulating the mode of cholesterol efflux and Aβ clearance in mice model [130].
Amyloid 42 aggregative prone peptides are the plaque forming peptides that are greatly associated with 7KCh where these molecules enhance the fibrillation of peptides [131]. The role of 7KCh in the cleavage of amyloid precursor protein and the formation of Aβ1–42 peptide was unclear, hence the activity of beta site APP cleavage enzyme which is responsible for the formation of aggregative amyloid peptide in 7KCh induced cells was analysed. Our analysis revealed that the BACE1 mRNA levels were elevated upon 7KCh induction due to which protein misfolds. Consequently, BACE1 upregulation 7KCh increased the level of HSP70 as well as ATF-6 the unfolded protein response and endoplasmic stress activators in our study, similar to those of prior evidence [132]. In contrast, bornyl acetate and menthol co-treatment suppressed the action of BACE1 levels and UPR sensor mRNA levels.
In addition, the antioxidant genes quinine oxidoreductase (NQO1), hemeoxygenase 1 (HO-1) and glutathione peroxidase (GPx) were observed with a marked level of downregulation by 7KCh which was consistent with our results showing downregulation of HO-1 and BCl2 genes [20]. Natural compounds like chlorogenic and resveratrol acid in previous review reported the upregulated the BCl2, HO-1 expression in PC-12 cells and SH-SY5Y cells respectively [133]. Supportingly, our results upon bornyl acetate and menthol treatment upregulated the anti-oxidant genes against 7KCh proving their potential anti-oxidant role.
Besides, 7KCh and its reflection on amyloid generation act as an instigator of inflammasome activation that upregulates the pro-inflammatory mediators such as TNF-α, IL-1β and NF-κB [126]. Dependably, our investigation focussed on the protein level expression of amyloidogenic markers BACE1 and Aβ1–42, pro-inflammatory as well as apoptotic markers iNOS, TNF-α, IL-1β, NF-κB, caspase-3 and abruptly showed the upregulated protein levels. Previously the mechanism of monoterpenes regarding AD pathways has been extensively studied with the compounds such as myrtenal, verbenone, carvacrol, 1,8-cineole (eucalyptol) and α-pinene, β-pinene, α-terpinene, γ-terpinene, 3-carene, limonene, sabinene, trans-anethole, thymohydroquinone, carvacrol, thymoquinone, thymol, linalool and pulegone exhibited therapeutic aspects of AD treatment [134]. Catalpol a monoterpenoid found to inhibit Aβ production profoundly through β-secretase inhibition, BDNF upregulation, anti-oxidant system to reduce free oxygen radical and pro-inflammatory mediators [135, 136]. Considering previous reports comparable results were obtained in our study in the co-treatment of 7KCh induced cells with bornyl acetate and menthol-mediated downregulation of transcription factors and amyloidogenic mediators.
In conclusion, this in vitro investigation states that the natural monoterpene compounds bornyl acetate and menthol are cytoprotective in nature, hence employed as a therapeutic agent in this study. Effects of bornyl acetate and menthol associated with lipid mediated inflammation and amyloidogenic pathway in 7-ketocholesterol-induced SH-SY5Y cells was examined. The lipid accumulation, free radical generation, amyloid fibril formation, intracellular calcium accumulation, mitochondrial, cellular membrane damage and oxidation of macromolecules were reduced by bornyl acetate or menthol co-treatement. Bornyl acetate and menthol upregulated the anti-oxidant genes HO-1, lipid regulatory ABCA1 expression and suppressed the TLR4 mediated lipid uptake, which further decreased the levels of pro-inflammatory (IL-6, iNOS) and pro-apoptotic (PARP-1, cathepsin D and caspase-3) mediators involved neurodegeneration. Activated amyloidogenic pathway by BACE1 activity and Aβ-42 levels induced by 7-ketocholesterol was evident from the results, which was significantly inhibited by bornyl acetate and menthol. From our in vitro investigation, we conclude that these monoterpenes as an efficient example of pharmacological element to suppress the devastating neurodegeneration. Further in vivo experimental analyses of several lipid associated targets and clinical trials would address them as an ideal medication for Alzheimer’s disease onset and progression without any side effects.