D-chiro-inositol increases antioxidant capacity and longevity of Caenorhabditis elegans via activating Nrf-2/SKN-1 and FOXO/DAF-16

D-chiro-inositol (DCI) is an isomer of inositol, abundant in many foods, such as beans and buckwheat, with insulin-sensitizing, anti-inflammatory, and antioxidant effects. DCI has been used to relieve insulin resistance in diabetes and polycystic ovary syndrome in combination with inositol or D-pinitol. Here, we investigated the effect of DCI on aging and stress resistance in C. elegans . We found that DCI could prolong the lifespan of C. elegans by up to 29.6 %. DCI significantly delayed the onset of neurodegenerative diseases in models of C. elegans . DCI decreased the accumulation of A β 1 – 42 , alpha-synuclein, and poly-glutamine, the pathological causes of Alzheimer's, Parkinson's, and Huntington's diseases, respectively. DCI significantly increased the stress resistances against pathogens, oxidants and heat shock. Moreover, D-chiro-inositol reduced the content of ROS and malondialdehyde by increasing the total antioxidant capacity and the activity of superoxide dismutase and catalase. Above effects of DCI requires the transcription factors FOXO/DAF-16 and Nrf-2/SKN-1. DCI also increased the expression of downstream genes regulated by FOXO/DAF-16 and Nrf-2/SKN-1. In conclusion, DCI enhanced the antioxidant capacity and healthy lifespan of C. elegans by activating DAF-16, SKN-1, and HSF-1. Our results showed that DCI could be a promising antiaging agent that is worth further research on the mech- anism and health supplemental application of DCI.


Introduction
D-chiro inositol (DCI) is an isomer of inositol that is abundant in beans, buckwheat, and other foods. DCI can be converted from myoinositol by NAD/NADH epimerase in an insulin dependent way (Pak et al., 1992). Inositol is a membrane molecule acting as a secondary messenger in signal transduction. DCI has steroidogenic activity and could improves sperm mitochondrial membrane potential (Condorelli et al., 2022). DCI has been used to relieve insulin resistance in diabetes and polycystic ovary syndrome (Simic et al., 2022). Specifically, DCI could improve metabolic disorders, such as reducing blood glucose levels in db/db mice, reducing liver lipid deposition, and reducing hyperlipidemia (Fan et al., 2020a;Fan et al., 2022;Yang et al., 2022). Metabolic disorders could induce stresses including oxidative stress and inflammation, subsequently cause damages, such as endothelial dysfunction, renal tissue damage, and cognitive decline. DCI could relieve these stresses and damages by activating the AdipoR-AMPKα/ PPARs pathway and inhibiting NF-κB protein expression (Fan et al., 2022;Yang et al., 2022;Zhang et al., 2017a;Zhao et al., 2018). A recent study reported that DCI could prolong longevity through insulin signaling (IIS) and autophagy pathways in fruit flies (Du et al., 2022a). Above researches showed that DCI have activities on steroidogenesis, metabolism, and inflammation. The physiological and therapeutic properties of DCI might not be fully discovered and is worth further investigation.
The nematode C. elegans is a classic model organism for the research of aging and neurodegenerative diseases (Alexander et al., 2014;Carretero et al., 2017;Pir et al., 2017;Schmeisser and Parker, 2018;Sulston et al., 1983;Zhang et al., 2017b). C. elegans has a transparent body and a short lifespan (about three weeks). C. elegans shares high genetic homology with mammals. The aging regulating pathways are highly conserved between C. elegans and mammals (including humans), such as the insulin/IGF-1 pathway, dietary restriction pathways, and the mTOR pathway. Upon being activated by ligands (such as insulin), the insulin receptor DAF-2 phosphorylates PI3K/AGE-1 and leads to subsequent phosphorylation of pyruvate dehydrogenase lipoamide kinase isozyme (PDK)-1, AKT-1/2, and serine/threonine-protein kinase (SGK)-1, which prevents the activation of FOXO transcription factor DAF-16. The lifespan of loss-of-function mutant of daf-2 and age-1 were doubled, while the loss-of-function mutants of akt-1/2 and sgk-1 were also long-lived. The activation of DAF-16 not only extends the lifespan, but also increases stress resistances of C. elegans. Since DCI could regulate metabolism and inflammation, we investigated the effect and potential mechanism of DCI on the aging and stress resistance of C. elegans.

Lifespan assay
When synchronized larvae have grown into L4 stage, worms were transferred to NGM plates containing DCI and FUDR (50 mg/mL). Worms were transferred to a new NGM plate daily until egg laying stops. Live worms were counted every day started from the tenth day of Body bend frequency of wild-type worms on day 5th and 10th after treatment with 400 μM of DCI. (E-F) Lipofuscin deposition in wild-type worms on day 10th after treated with 400 μM of DCI. Lifespan analysis was performed by using the SPSS package and Kaplan-Meier. The p-values were calculated with log-rank test. Data were expressed as the mean ± SD. It was considered statistically significant when p < 0.05. adulthood.

Aging-related phenotype analysis
Worms were maintained as described in the lifespan assay. For the body bending assay, the body bending of worms was counted for 20 s on the 5th day and 7th day of DCI treatment. For the lipofuscin accumulation assay, the intestine fluorescence of nematode was photographed on the 10th day of DCI treatment by using fluorescence microscopy (Leica DFC 7000T).

Stress resistance assays
For oxidative stress assay, the worms treated with DCI for 7 days from L4 larvae stage were transferred to NGM plates containing paraquat (20 mM, Sigma). The death of worms was counted daily. For heat shock assay, the worms treated with DCI for 7 days from L4 larvae stage were incubated under the ambient temperature of 35 • C for heat shock. The dead worms were counted every 1 h until all were dead. For pathogen resistance assay, worms treated with DCI for 7 days from L4 larvae stage were transferred to NGM plates containing Pseudomonas aeruginosa (PA14). The dead worms were counted daily until all were dead.

Reactive oxygen species (ROS) assay
L4 stage worms were spread on NGM plates containing DCI, paraquat (20 mM), or N-Acetyl-L-cysteine (NAC, 1 mM) and incubated for 7 days. After that, worms were collected and washed with M9 buffer. Then worms were incubated with fluorescent probe DCFH-DA (50 μM) and shaken in dark for 60 min at 35 • C. The levels of ROS were observed under a fluorescent microscope (Leica DFC 7000T).

Oxidative stress indicator assays
Worms treated with DCI for 7 days from L4 larvae stage were transferred to NGM solid medium containing 20 mM of methyl viologen and cultured for one day. Then, worms were collected and frozen at − 20 • C for 30 min. Next, worms were crushed and centrifuged at 12,000 ×g rpm/min for 10 min. The supernatant was collected. The protein concentration of the supernatant was determined by using the total Protein assay Kit (with standard: BCA Method). The activities of CAT, SOD, and T-AOC and MDA levels were measured according to the instruction of the kit.

DAF-16::GFP translocation assay
The transgenic strain TJ356 expresses DAF-16 conjugated with GFP (Henderson and Johnson, 2001). TJ356 worms at L4 stage were laid on plates containing DCI as the experimental group. The remaining worms of the same period were laid on plates without DCI as the control group. Half of the worms from the control group were incubated at 35 • C for 30 min and was further grouped as positive control. The untreated worms from control group were regrouped into negative control. Then the localization of DAF-16::GFP was observed under fluorescence microscope (Leica DFC 7000T). Each group includes at least 30 worms. The experiment was repeated three times independently.

Neurodegenerative diseases assay
The worms of transgenic strain CL4176 will express human amyloidβ protein in muscle when incubated at the elevated temperature of 23 • C and then become paralyzed (Link et al., 2003;Sadananda et al., 2021). The L1 CL4176 worms were incubated on NGM at 15 • C for 36 h (Xu et al., 2019). The NGM plates were then transferred to an incubator that was set to 25 • C. Paralyzed worms were counted every 1 h for the next 24 h. If CL4176 strains were found to be rigid and unable to perform normal oscillations, and the nematodes were defined as paralyzed.
The CL2006 worms expresses human Aβ 1-42 peptides in body wall muscle cells and was used as research model of AD (McColl et al., 2009). The CL2006 worms were treated as described in the previous lifespan assay. Then, the paralysis of worms was monitored on the tenth day until the worm was fully paralyzed. Each experiment includes at least 70 worms. The experiment was repeated three times.
The strain NL5901 worms expressing human ⍺-synuclein conjugated with yellow fluorescent protein (YFP) in muscle cells is commonly used as the nematode model of Parkinson's disease (Van Ham et al., 2008). NL5901 worms at L4 stage were treated with DCI. Then, body bending behavior analysis was performed on day 5th and 10th of DCI treatment. The fluorescent pictures of worms were taken on day 7th of DCI treatment. The strain BZ555 worms express GFP in dopaminergic neurons (Chalorak et al., 2021). BZ555 worms at L4 stage were washed into a centrifuge tube (1.5 mL) filled with 1 mL of NGM liquid M9 medium containing 10 mM of ascorbic acid, 50 μM of 6-hydroxydopamine, and 100 μL OP50 E. coli, and shaken at 20 • C for 1 h. Then, worms were collected and allocated to NGM plates without treatment (negative control group), or containing DCI, or containing 20 mM of levodopa (positive group) and incubated for 72 h at 20 • C. Finally, the profile of dopaminergic neurons in the head of worm was observed by fluorescence microscopy (Leica DFC 7000T). Each experimental group includes at least 30 worms. The experiment was repeated three times independently.
The transgenic strain AM141 (unc-54p::Q40::YFP) expresses Q40:: YFP in body wall muscle by the time it reaches adulthood and is used as a model of Huntington Disease (Morley et al., 2002;Gidalevitz et al., 2006). The synchronized L4 stage larvae of strain AM141 was transferred to NGM plates containing DCI and incubated at 20 • C. The accumulation of poly-glutamine (Poly-Q) was counted by fluorescence microscope (Leica DFC 7000T) every 24 h for 7 days. Each experimental group includes at least 30 worms. The experiment was repeated three times.

Protein expression quantification assay
Worms expressing fluorescent proteins were synchronized and raised to the L4 stage, then treated with DCI and incubated at 20 • C for 7 days before being photographed by fluorescence microscope (Leica DFC 7000T). Strain BC12921 was photographed at day 3 and day 7 of incubation. Each experimental group includes at least 30 worms. Each experiment was repeated three times.

The mRNA expression quantification through real-time quantitative PCR
N2 wild-type worms were incubated on NGM plates with or without DCI at 20 • C. Total RNA was isolated by using the Steadypure Universal RNA Extraction Kit (Accurate Biology). The total RNA was then reversetranscribed into cDNA by using PrimeScriptTM RT reagent Kit. The mRNA expression was quantified by SYBR Green Premix Pro Taq HS qPCR Kit (Rox Plus) on QuantStudio 6 Flex system. The relative mRNA expression levels of genes were calculated using the 2 -ΔΔCT method and normalized to the expression of gene cdc-42 (Zheng et al., 2017). The primer sequences are available in Supplementary Table S1.

Statistical analysis
Statistical analyses were performed by using SPSS Statistics and GraphPad Prism 6.02 software. Lifespan experiments were analyzed using Kaplan-Meier survival analysis. And other data were expressed as the mean ± SD. The p values were determined by two-tailed t-test. A p value < 0.05 was considered as a significant difference.

DCI extends the lifespan and slows the aging-related phenotypes of C. elegans
To investigate whether DCI could prolong the lifespan of wild-type C. elegans, we treated worms with different concentrations of DCI (viz. 50,100,200,400,and 800 μM) at 20 • C. We found that DCI could prolong the lifespan of wild-type worms at various concentrations. The most effective dosage of DCI at 400 μM prolonged the lifespan of worms by up to 29.62 % compared to untreated worms ( Fig. 1B and C, Table S2).
The ideal scenario for lifespan extension is increased healthy lifespan (Teo et al., 2020). The motility of worms decreases with aging (Huang et al., 2004). We therefore examined body bending behavior and lipofuscin accumulation in worms to assess whether DCI affects the healthy lifespan of worms. The results showed that DCI significantly enhanced body bending behavior and reduced lipofuscin accumulation in worms ( Fig. 1D-F, Tables S3 and S4).

DCI delays the development of neurodegenerative diseases in models of C. elegans
Alzheimer's disease (AD) was supposed to be caused by the toxic aggregation of amyloid beta peptide (Aβ) and abnormally phosphorylated tau (Jagust, 2018). The worms strains CL4176 and CL2006 were used as disease model to investigate whether DCI has an effect on Alzheimer's disease. When cultured at 25 • C, worms CL4176 expresses Lifespan analysis was performed using the SPSS package and Kaplan-Meier. The p-values were calculated using log-rank test. Data were expressed as the mean ± SD. It was considered statistically significant when p < 0.05. human Aβ1-42 in the cytoplasm of body wall muscle cells and then become paralyzed (Lublin and Link, 2013). Our results showed that DCI delayed the progress of paralysis by an average of 13.86 % (p < 0.001, Fig. 2A, Table S8). CL2006 worms express human A β1-42 peptide in body wall muscle at 20 • C (Meng et al., 2013). The adults of CL2006 worms present progressive paralysis. We found that DCI could delay the paralysis rate of CL2006 by a mean of 14.47 % (p < 0.001, Fig. 2B, Table S8).
The aggregation of α-synuclein and the atrophy of black dopaminergic (DA) neurons lead to movement defect in Parkinson's disease (PD). The strain NL5901 expresses α-synuclein protein fused with YFP in body wall muscle cells was used as PD model. We found that DCI both delayed the age-related decrease in motility of the worms NL5901 (p < 0.001, Fig. 2D, Table S3) and the intensity of YFP fluorescence by about 35.82 % (p < 0.0001, Fig. 2C, Table S9). The worms BZ555 expresses GFP in dopaminergic neurons. So the degeneration of the DA neurons in worms BZ555 by a liquid medium containing 50 mM of 6-OHDA could be determined by GFP fluorescent photograph. Our results showed that DCI treatment significantly prevented the damage of DA neurons compared with the negative control group (p < 0.001, Fig. 2E-F, Table S9), although slightly inferior to the positive drug levodopa.
The aggregation of polyglutamine causes progressive lesions in basal ganglia, and the clinical symptoms of Huntington's disease (HD), such as abnormal movement and cognition. The AM141 worms expressing polyglutamine fused with YFP was used as the model of HD. We found that DCI could significantly suppress the aggregation of Q40::YFP (p < 0.001, Fig. 2G-H, Table S10). Above results showed that DCI could significantly delay the onset of neurodegenerative disease in models of C. elegans.

DCI enhances the heat stress resistance of C. elegans via activating HSF-1
Increased stress resistance of worms is a phenotypic indicator of longevity (Kenyon, 2010). Therefore, we examined effect of DCI on heat stress, oxidative stress, and pathogenic stress resistance in C. elegans. Our results showed that worms treated with DCI for 7 days lived an average of 47.28 % longer than untreated worms under heat stress of 35 • C (p < 0.001, Fig. 3A, Table S5). In oxidative stress assay, our results showed that DCI extend the lifespan of wild-type worms under acute oxidative stress (20 mM of paraquat) by about 18.97 % (p < 0.001, Fig. 3B, Table S6). In the case of pathogenic stress containing Pseudomonas aeruginosa (PA14), the lifespan of worms treated with DCI was extended by an average of 14.48 % (p < 0.001, Fig. 3C, Table S7).
Heat shock factor 1 (HSF-1) is a key transcription factor regulating heat stress and protein folding homeostasis. Therefore, we examined the expression of hsf-1 and its downstream target genes: hsp-6, hsp-16.2, and hsp-60. Our results showed that DCI significantly increased the mRNA expression levels of the above genes in wild type worms, but not in the loss-of-function mutant hsf-1(sy441) (Fig. 3D, Table S12). We then examined effect of DCI on the expression of the three heat shock proteins conjugated with fluorescent proteins HSP-4::GFP, HSP-60::GFP and HSP-6::GFP in the strains SJ4005 (hsp-4::GFP), SJ4058 (hsp-60::GFP), and SJ4100 (hsp-6::GFP), respectively. The results showed that DCI increased the expression of all three proteins ( Fig. 3E-J, Table S9). We also showed that DCI did not extend the lifespan of the hsf-1 deletion mutant (Fig. 3K, Table S8). Above results indicated that DCI requires HSF-1 to increase the heat stress resistance and extend the lifespan of C. elegans.

DCI enhances the antioxidative activity of C. elegans
Since DCI could extend the lifespan of worms under acute oxidative stress, we determined the content of ROS in C. elegans after DCI treatment. Our results showed that DCI significantly reduced the ROS levels in C. elegans (Fig. 4A-B, Table S11). Malondialdehyde (MDA) is an important metabolite produced by oxygen radicals in living organisms. MDA is cytotoxic for it attacking unsaturated fatty acids in lipids and is often used as one of the main markers of oxidative damage (Esterbauer and Cheeseman, 1990). The malondialdehyde content in the worms was significantly reduced after DCI treatment compared to the control group (Fig. 4C). Above results show that DCI can delay the oxidative damage suffered by C. elegans.
Antioxidant capacity of worms increases when antioxidant system was activated (Lin et al., 2020). We examined the total antioxidant capacity (T-AOC) and the activity of Superoxide Dismutase (SOD) and Catalase (CAT) in worms treated with DCI. The results showed that DCI significantly increased T-AOC and the activity of SOD and CAT (Fig. 4D-F). We found that DCI increased the mRNA expression levels of genes sod-3, ctl-2 and gst-4 (Fig. 4G, Table S12). We also examined the protein expression level of SOD-3 and showed that DCI significantly enhanced the expression level of SOD-3::GFP ( Fig. 4H-I, Table S9).
The transcription factor Nrf-2/SKN-1 is the essential regulator of antioxidant activity and xenobiotic defense. Upon activation, the expression of SKN-1 would accumulate in the nucleus. So we determined the effect of DCI on the fluorescence intensity and distribution of skn-1b/ c::GFP. We found that DCI could not increase the total expression of skn-1b/c::GFP, but increased nuclear ectopia of skn-1b/c::GFP by about 33 % (Fig. 4J, Table S9, Fig. 4K). DCI also could no increase the mRNA expression level of skn-1 (Fig. 4G, Table S12), while the oxidative stress did increase the mRNA expression level of skn-1 (Fig. 4G, Table S12). To further examine whether the increased antioxidative capacity induced by DCI requires SKN-1, we determined the mRNA levels of genes involved in antioxidative activity in worms with deletion of skn-1. Our results showed that DCI could not increase the mRNA levels of genes sod-3, ctl-2, and gst-4 in mutant deleted with skn-1 (Fig. 4G, Table S12). We also examined the lifespan of the skn-1deletion mutant EU1 and showed that DCI could not extend the lifespan of the skn-1 mutant (Fig. 4L, Table S8).

DCI requires the transcription factor FOXO/DAF-16 to extend the lifespan of C. elegans
The transcription factor FOXO/DAF-16 acts as downstream target of IIS/IGF-1 pathway, is the central regulator for development, reproduction, metabolism, stress response, and lifespan. When disinhibited from AKT-1/2, DAF-16 translocate into nucleus and initiate the expression of targeted genes. Here we showed that DCI significantly increased the aggregation of DAF-16 in nucleus (p < 0.001, Fig. 5A-B). Furthermore, DCI decreased the mRNA level of pdk-1 and increased the mRNA levels of daf-16 and its targeted gene sod-3 (p < 0.001, Fig. 4G and 5E, Table S12). DCI also increased the protein level of SOD-3 (Fig. 4I, Table S9). We then examined the lifespan of daf-16 and akt-2 mutant strains and showed that DCI could not extend the lifespan of the loss-offunction mutants from daf-16 and akt-2 (p < 0.001, Fig. 5C-D, Table S8).

The effect of DCI on autophagy and energy sensing signaling in C. elegans
Autophagy is a cellular recycling process degrading xenobiotic and unfolded proteins and has an important role in cellular homeostasis, metabolism, and lifespan. Worms BC12921 express autophagy substrate SQST-1/p62 tagged with GFP (Tian et al., 2010). The decrease of autophagy substrates P62/SQST-1 could be inferred from the intensity of GFP and could be indicated as the increased activity of autophagy. We found that there was no significant difference of the fluorescence intensity in BC12921 between DCI treatment and the corresponding untreated worms (Fig. 6A-B, Table S9). We measured the mRNA expression levels of the autophagy genes (bec-1 and lgg-1) and showed that DCI did not increase the mRNA levels of bec-1 and lgg-1 genes (Fig. 6C, Table S12). Moreover, DCI extended the lifespan of the loss-offunction mutant of autophagy gene atg-18 by about 13.33 % (Fig. 6D,

hsf-1 h s f -1 h s p -6 h s p -6 0 h s p -1 6 . 2
hsf-1(sy441) hsf-1(sy441) and Kaplan-Meier. The p-values were calculated using log-rank test and considered statistically significant when p < 0.05. Data were expressed as the mean ± SD. The p-values were calculated using log-rank test and was considered statistically significant when p < 0.05. Data were expressed as the mean ± SD. Table S8). Above results indicated that DCI did not activate autophagy to extend the lifespan. The NAD + -dependent protein deacetylases SIR-2.1 is a member of the Sirtuins family and plays important roles in transcriptional repression, DNA damage repair, stress response, and aging (Greiss et al., 2008). Overexpression of sir-2.1 could extend the lifespan of C. elegans. Sir-2.1 is required for lifespan extension of dietary restriction in eat-2 worms. Here we showed that DCI could increase the mRNA level of sir- Lifespan analysis was performed using the SPSS package and Kaplan-Meier. The p-values were calculated using log-rank test and was considered statistically significant when p < 0.05. The data were expressed as the mean ± SD.

akt-2 daf-16 s i r -2 . 1 p d k -1 d a f
2.1 (Fig. 5E, Table S12). The gene aak-2 encoding the alpha subunit of AMP-activated protein kinase (AMPK), which senses and regulates cellular energy homeostasis, stress resistance, and lifespan. We therefore examined the expression of aak-2 and found that the mRNA level of aak-2 was significantly upregulated in DCI-treated wild-type C. elegans (Fig. 6C, Table S12).

Discussion
In the present study, we showed that exogenous addition of DCI significantly extended the lifespan of C. elegans. DCI enhanced body bending behavior and reduced lipofuscin accumulation in worms. DCI significantly delayed the onset of neurodegenerative diseases and their pathological deterioration in models of C. elegans. The stress resistance of C. elegans to heat, oxidants, and pathogens was also increased by DCI. The total anti-oxidative activity and the activity of anti-oxidant enzymes in C. elegans were enhanced with treatment of DCI. The activation of the transcription factors HSF-1, SKN-1, and DAF-16 by DCI might account for enhanced stress resistance and delayed aging in C. elegans. Above results suggest that DCI has good anti-aging and antioxidant effects and could be a potential source for the development of novel therapeutic agents for the treatment of aging and an adjuvant therapeutic agent for aging-related diseases.
Aging is associated with increased stresses such as mitochondrial dysfunction, DNA damage, protein misfolding and aggregation, which result in increased ROS, decreased energy efficiency, and physiological decline (López-Otín et al., 2013;Bou-Teen et al., 2021). Increased stress resistance could extend lifespan. Many studies have shown that delaying C. elegans aging is associated with enhanced and improved resistance to oxidative stress (Qu et al., 2022;Deng et al., 2022). DCI can reduce the levels of ROS and MDA and increase the levels of SOD, CAT and T-AOC in wild-type worms (Fig. 4B-F). DCI activates the crucial oxidative stress regulator Nrf-2/SKN-1 and required Nrf-2/SKN-1 to prolong the lifespan of C. elegans (Fig. 4L). DCI also increased heat stress resistance by activating HSF-1. DAF-16 regulates the activity of Nrf-2/SKN-1 and HSF-1, is the central regulator for stress responses such as immune response, oxidative response, heat shock response, and protein homeostasis. DAF-16 is activated and required for lifespan extension treated with DCI in C. elegans. Overall, above results suggest that DCI extends the lifespan and delays the progression of neurodegenerative diseases in models of C. elegans by increasing the stress resistances, especially the antioxidative capacity.
DCI has been reported to relieve insulin resistance and improve metabolic disorders (Carretero et al., 2017;Kenyon, 2010). DCI could improve glucose metabolism by upregulating IRS2, PI3K, AKT, and GLUT4 (Fan et al., 2020a). In C. elegans, worms with loss-of-function mutation of insulin receptor were long-lived (Zhu et al., 2020;Lapierre and Hansen, 2012). Insulin receptor negatively regulates the activity of DAF-16 through cascade kinase activities. Our results showed that DCI requires and activates daf-16 to extend the lifespan of C. elegans. Consistent with the reports from mammals (Yang et al., 2022;Zhang et al., 2017a;Zhao et al., 2018), our results showed that DCI could activate AMPK, suggesting that DCI might act to improve the metabolic status of C. elegans, rather than sensitizing the insulin pathway. DCI prolonged the lifespan of the worms with mutation of autophagy gene atg-18. Our results also showed that DCI could not activate the activity of autophagy. These results suggest that DCI might not affect the lifespan of C. elegans through the autophagic pathway. However, a recent study reported that DCI could extend the lifespan of drosophila through activating insulin and the autophagy signaling pathway (Du et al., 2022b). This might be caused by the presence of species-specific mechanisms (Berkel and Cacan, 2021). It is an interesting direction to explore the specific reasons for these differences.
Both DCI and myo-Inositol (MI) are effective in sensitizing insulin, and in preventing and treating metabolic and reproductive disorders and male fertility disturbances (Dinicola et al., 2021a). There are nine different stereoisomers of inositol, but about 99 % of inositol in mammals are myo-Inositol (MI) . MI enhances glucose uptake by promoting GLUT translocation to the plasma membrane, inhibits adenylate cyclase, and reduces free fatty acid release from adipose tissue (Ijuin and Takenawa, 2012). MI acts as an FSH second messenger to regulate the proliferation and maturation of granulosa cells and anti-Mullerian hormone (AMH) production (Milewska et al., 2016). DCI reduces the expression of aromatase, the key steroidogenic enzymes genes CYP19A1 and P450scc, and insulin-like growth factor 1 receptor (IGF-1R), subsequently stimulating the ovarian production of androgens and inhibiting the synthesis of estrogens (Sacchi et al., 2016). DCI stimulates glycogen synthase (Fan et al., 2020b) and stimulates pyruvate dehydrogenase, increasing the production of adenosine triphosphate (ATP) (Heimark et al., 2014). Insulin regulates the transformation of MI to DCI through an epimerase (Monastra et al., 2021). Above discoveries indicate that there exist complex feedback regulations among insulin, MI, and DCI. Therefore, the ratio between MI and DCI (M/D) in each tissue or organ is specific and highly preserved to ensure their healthy state and proper functionality (Dinicola et al., 2021a). Altered inositol ratios may account for pathological conditions, while maintaining a healthy ratio could have recovering effects (Heimark et al., 2014). It is interesting to investigate the effect of DCI on the aging of mammals.

Conclusion
Our data demonstrate that D-chiro-inositol, extractable from foods like legumes and buckwheat, enhances the antioxidant capacity and healthy lifespan of C. elegans by activating DAF-16, SKN-1/NRF-2, and HSF-1. DCI is clinically effective as an insulin sensitizer, in treating metabolic and reproductive disorders, and in treating male fertility disturbances (Dinicola et al., 2021b;De Luca et al., 2021;Ashu et al., 2020;Xin et al., 2021;Bahadur et al., 2021). It is worth continuing the discovery of beneficial effects and mechanisms of DCI in health applications.

Funding
This work was supported by grants from the National Natural Science Foundation of China (82171555), Central Nervous System Drug Key Laboratory of Sichuan Province (230003-01SZ), and Luzhou Science and Technology Program (2022SWMU4).

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
No data was used for the research described in the article.

Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.exger.2023.112145.