Fig 1: The neuroprotective effects of D-allose.

(A) Neurological function was assessed using the mNSS scale at 24 and 48 hours after ischemia-reperfusion (n=9).

(B) Cerebral infarct volumes were measured using TTC staining at 24 and 48 hours after reperfusion with TTC (n=9).

(C) Brain edema was measured at 24 and 48 hours after reperfusion (n=9).

(D) Assessing neurological function after treatment (n=9).

(E) D-allose was administered by intraperitoneal injection within 5 minutes after reperfusion. TTC staining was performed to evaluate the infarct volumes after treatment (n=12).

(F) Brain edema was measured after treatment (n=12).

(G) Assay of apoptosis-related molecules by Western blot (n=12).

(H) Detection of apoptosis in neuronal cells of mice after being MCAO by TUNEL assay (n=12).

Levels of TNF-α（I）, IL-1β(J) and IL-6(K) in cerebral tissue were detected by ELISA kits (n=12).

**P ＜0.01 compared with Sham group;^nsP ＞0.05, ^##P ＜0.01 compared with MCAO group.

Fig 2: Effects of D-allose on the cytotoxicity and apoptosis induced by OGD/R in HT22 cells.

(A) HT22 cells were subjected to OGD damage at different time lengths and cell viability was detected by CCK8 assay.

(B) HT22 cells were subjected to OGD/R injury and LDH levels were measured.

(C) Cell viability was detected by CCK8 assay after administration of D-allose treatment.

(D) Detection of LDH levels after D-allose treatment.

(E) Effects of D-allose on the expression levels of Bax, Bcl-2, Caspase 3, and Caspase 1in HT22 cells subjected to OGD/R.

(F) Effects of D-allose on HT22 cells apoptosis induced by OGD/R, stained with TUNEL.

The effects of D-allose on the levels of TNF-α (G), IL-1β (H) and IL-6 (I) were measured by ELISA kits.

**P ＜0.01 compared with Con group;^##P ＜0.01 compared with OGD/R group.

Fig 3: Increased expression of Gal-3 in ischemic/reperfusion(I/R) injury in vivo and in vitro

(A) Changes in Gal-3 mRNA levels in brain tissue at 24 and 48 hours after MCAO was measured by RT-PCR (n=9).

(B) Expression of Gal-3 protein content in brain tissue at 24 and 48 hours after MCAO was measured by Western blot (n=9).

(C) Observation of Gal-3 expression in brain tissue at 24 and 48 h post-MCAO by immunofluorescence (n=9).

(D) Detection of Gal-3 mRNA expression in HT22 cells after different lengths of OGD/R injury using RT-PCR.

(E) Examination of Gal-3 expression in HT22 cells after different durations of OGD/R injury by using Western blot.

(F) Observation of Gal-3 expression in HT22 cells after different durations of OGD/R injury as measured using immunofluorescence.

(G) Using IF to observe lentivirus transfection.

(H) Detection of mRNA expression of Gal-3 after lentiviral transfection.

(I) Detection of protein expression levels of Gal-3 after transfection with lentivirus.

(J) CCK8 assay to detect cell viability after transfection with lentivirus.

(K) Detection of LDH levels after the transfection of lentivirus

(L) Western blot detection of caspase 3, Bax, Bcl-2, and caspase 1 proteins expression after LV transfection.

(M) TUNEL staining was used to observe the apoptosis of HT22 cells after transfection with different lentiviruses.

(N)-(P) The expression levels of inflammatory factors in HT22 cells transfected with lentivirus and then damaged by OGD/R were measured using ELISA kits.

**P ＜0.01 compared with Con group; ;^nsP ＞0.05, ^##P ＜0.01 compared with OGD/R group.

Fig 4: Gal-3 directly combined with TLR4 in vitro

(A) Immunofluorescence observation of co-localization between Gal-3 and TLR4 on HT22 cells.

(B) An immunofluorescent observation of the co-localization of Gal-3, neurons, and TLR4 in brain tissue (n=4).

(C) Analysis of the protein structure using 3D structure prediction techniques. A mock structural map of the binding of Gal-3 (cyan) to TLR4 (green) was obtained, as well as the specific sites where the two bind.

(D) Immunoblot showing the presence of Gal-3 and TLR4 in an immune complex formed after the pull-down of TLR4 and Gal-3 in the con group and OGD group of HT22.

Fig 5: Gal-3 activate TLR4/ PI3K/AKT signaling axis in vitro

(A) Western blot detection of the expression of related proteins in the Gal-3-TLR4-PI3K-AKT pathway after administration of D-allose treatment.

(B) Expression of related proteins in the Gal-3-TLR4-PI3K-AKT pathway after lentivirus transfection detected by Western blot.

(C) Western blot to detect the expression of signaling pathways and apoptosis-related proteins after transfection with Gal-3 lentivirus and administration of agonists and inhibitors of TLR4.

(D) Detection of HT22 cell viability by CCK8 assay after transfection with Gal-3 lentivirus and administration of agonists and inhibitors of TLR4.

(E) Measurement of LDH levels in HT22 cells.

(F) TUNEL staining was used to observe apoptosis in each group of HT22 cells.

(G)-(I) Use of ELISA kits to detect the secretion level of inflammatory factors in all groups of cells.

**P ＜0.01 compared with Con group; ;^nsP ＞0.05 ,^##P ＜0.01 compared with OGD/R group;^&&P ＜0.01 compared with OGD/R+sh-Gal-3 group.

Fig 6: D-allose regulated TLR4/ PI3K/AKT pathway through Gal-3 to protect HT22 cells from OGD/R-induced injury

(A) Detection of HT22 cell viability after transfection with LV and D-allose treatment by CCK8 assay.

(B) Measurement of LDH levels in HT22 cells after transfection with lentivirus and D-allose treatment.

(C), (D), (E) Assay of relevant inflammatory factors in each group of HT22 cells by ELISA kit.

(F) Detection of apoptotic proteins such as Bax and related proteins in the Gal-3-TLR4 signaling pathway by Western blot.

(G) TUNEL staining to observe apoptotic cells in each set of cells.

**P ＜0.01 compared with Con group;^##P ＜0.01 compared with OGD/R group;^nsP ＞0.05, ^&&P ＜0.01 compared with OGD/R+D-allose group; ^$P ＜0.05 ,^$$P ＜0.01 compared with OGD/R+sh-NC group.

Fig 7: D-allose inhibit TLR4/ PI3K/AKT signaling to attenuates neuroinflammation and apoptosis via inhibiting Gal-3 after MCAO/R injury

(A) RT-PCR technique to detect the mRNA expression level of Gal-3 in mice after transfection with AAV (n=9).

(B) Western blot was performed to detect the protein expression level of Gal-3 in mice after AAV transfection (n=9).

(C) Detection of fluorescence display in mice transfected with AAV by immunofluorescence (n=9).

(D) The successfully transfected mice were subjected to MCAO injury and given D-allose treatment, and their neurological function was scored (n=12).

(E) TTC staining was used to observe the change in infarct volume (n=12).

(F) Brain edema was observed in each group of mice after the injury (n=12).

(G) Western blot was performed to detect the expression of Gal-3-TLR4 signaling pathway and apoptosis proteins in every group of mice (n=12).

(H) TUNEL staining to observe the apoptosis of neurons in mouse brain tissue (n=12).

(I) (J) (K)ELISA kits were used to detect the secretion levels of TNF-α, IL-1β and IL-6 in brain tissue (n=12).

^nsP ＞0.05, **P ＜0.01compared with Sham group; ^##P ＜0.01 compared with MCAO group;^nsP ＞0.05, ^&P ＜0.05,^&&P ＜0.01 compared with MCAO+D-allose group.

Fig 8: Potential mechanisms for the neuroprotective effects of D-allose on I/R-induced brain injury in mice and HT22 cells. D-allose protects the brain from ischemia-reperfusion-induced apoptosis and inflammation by suppressing Gal-3 expression and transcriptional processes that inhibit TLR4 and activate its mediated PI3K/AKT phosphorylation.