PMC

Intraperitoneal injection of curcumin (200 μg/g BW) in P7 mice at 60 min or 120 min post-HI significantly reduces tissue damage, cell death and glial response. (A) Curcumin treatment at 60 min post HI (n = 8) reduced volume loss compared to HI controls (n = 10) with significant, individual decrease (Kruskal-Wallis test with Bonferroni correction) in cortex (p = 0.036), hippocampus (p = 0.007), striatum (p = 0.003), thalamus (p = 0.005), and overall volume loss (p = 0.002). However, no main effect of the treatment was observed. No differences were registered between curcumin and DMSO (n = 7) treated littermates. (B) Administration of curcumin at 120 min post HI (n = 7) surprisingly increased the level of tissue loss however no significant main effect of treatment was observed. Pairwise comparison (one-way ANOVA with Bonferroni correction) revealed significant increase of volume loss in cortex in curcumin treated compared to HI (n = 7, p = 0.05) and to DMSO-treated littermates (n = 7, p = 0.017). (C) Curcumin treatment at 60 min post HI reduced TUNEL+ cell death compared to HI- and DMSO-treated littermates (Kruskal-Wallis test). Pairwise comparison (Bonferroni correction) revealed significant decrease of TUNEL+ cell death in curcumin treated compared to HI animals in cortex (p = 0.064), pyriform cortex (p = 0.064), hippocampus (p = 0.003) and overall (p = 0.001). Pairwise comparison (Bonferroni correction) revealed significant decrease of TUNEL+ cell death in curcumin compared to DMSO-treated animals in cortex (p = 0.003), pyriform cortex (p = 0.013), hippocampus (p = 0.042), and overall (p = 0.012). (D) Administration of curcumin at 120 min post HI reduced TUNEL+ cell death compared to HI- and DMSO-treated littermates (tone-way ANOVA, main effect p = 0.001). Pairwise comparison (Bonferroni correction) revealed significant decrease of TUNEL+ cell death in curcumin treated compared to HI animals in hippocampus (p = 0.002), striatum (p = 0.02), and overall (p = 0.001). Pairwise comparison (Bonferroni correction) revealed significant decrease of TUNEL+ cell death in curcumin compared to DMSO-treated littermates in hippocampus (p = 0.012), striatum (p = 0.022) and overall (p = 0.002). (E) Administration of curcumin at 60 min post HI reduced microglial activation compared to DMSO-treated littermates and HI controls, although the main effect of treatment did not reach significance. However, compared to HI controls, curcumin treatment reduced microglial activation with significant, individual decrease (Kruskal-Wallis test with Bonferroni correction) in external capsule (p = 0.042). Compared to DMSO-treated littermates curcumin treatment at 60 min post-HI reduced microglial activation with significant, individual decrease (Kruskal-Wallis test with Bonferroni correction) in cortex (p = 0.028). (F) Administration of curcumin at 120 min post HI reduced microglial activation compared to HI- and DMSO-treated littermates (Kruskal-Wallis test, main effect p = 0.002). Pairwise comparison (Bonferroni correction) revealed significant decrease of microglia activation in curcumin treated compared to HI animals in pyriform cortex (p = 0.03), hippocampus (p = 0.002), striatum (p = 0.002), thalamus (p = 0.01), external capsule (p = 0.006) and overall (p = 0.004). Pairwise comparison (Bonferroni correction) revealed significant decrease of microglia activation in curcumin compared to DMSO-treated littermates in pyriform cortex (p = 0.018), external capsule (p = 0.022), and overall (p = 0.016). (G) Curcumin treatment at 60 min post HI did not affect ipsilateral reactive astrogliosis. (H) Administration of curcumin at 120 min post HI reduced ipsilateral reactive astrogliosis (Kruskal-Wallis test with Bonferroni correction) in cortex compared to HI (p = 0.049) and DMSO-treated littermates (p = 0.006), however no main effect of the treatment was observed. (*p < 0.05). Abbreviations: CTX, cerebral cortex; EC – external capsule; HIP, hippocampus; PYR, pyriform cortex; STR, striatum; THAL, thalamus.

Intraperitoneal injection of curcumin in P7 mice post-HI reduces ipsilateral volume loss in males, females and combined (males + females), decreases myelin loss in males, but does not provide functional protection assessed through slipping test. (A) Intraperitoneal injection of 200 μg/g curcumin straight after HI decreased tissue loss at 21 days post HI (P28) in males compared to DMSO-treated littermates and untreated HI controls (curcumin n = 11, DMSO n = 8, HI n = 9; curcumin vs. HI p = 0.0017, curcumin vs. DMSO p = 0.0003). (B) Curcumin treatment at 200 μg/g straight after HI did not affect tissue loss at 21 days post HI in females compared to DMSO-treated littermates or to untreated HI controls. (curcumin n = 8, DMSO n = 7, HI n = 8). (C) The levels of tissue loss in the curcumin treated animals decreased compared to DMSO-treated littermates and untreated HI controls (curcumin n = 19, DMSO n = 15, HI n = 17; curcumin vs. HI p = 0.0024, curcumin vs. DMSO p = 0.0014). (D) The level of tissue loss observed in females was lower compared to the males, however the differences did not reach significant values (p = 0.085). This suggests higher susceptibility of the males to HI insult. (E–G) Subregional assessment of tissue loss at 21 days post HI in males (E, curcumin n = 11, DMSO n = 8, HI n = 9), females (F, curcumin n = 8, DMSO n = 7, HI n = 8) and combined (males + females) (G, curcumin n = 19, DMSO n = 15, HI n = 17). Curcumin treatment with 200 μg/g immediately post HI resulted in a reduction of subregional tissue loss in all studied regions in males (E) and combined (males + females) (G) with significant differences observed in thalamus (males p = 0.0292, combined (males + females) p = 0.0242). (F) Curcumin treatment had no effect on subregional differences in tissue loss in females. (H–J) MBP immunoreactivity at 21 days post HI (P28). Quantification of the ipsilateral external capsule, striatum, and overall in optical luminosity values (OLV, Mean + SEM). (H) In males curcumin treatment with 200 μg/g immediately post HI resulted in increased levels of MBP immunoreactivity compared to untreated HI controls (curcumin n = 11, DMSO n = 8, HI n = 9), with significant differences in striatum (Two-way ANOVA with post hoc Tukey’s test, p = 0.042). In females (I, curcumin n = 8, DMSO n = 7, HI n = 8) and combined (males + females) (J, curcumin n = 19, DMSO n = 15, HI n = 17), immediate treatment with 200 μg/g curcumin had no effect on MBP immunoreactivity. (K–M) Curcumin treatment with 200 μg/g immediately post HI did not affect the number of missed steps (slipping test) at 21 days (P28) post HI in males (K, curcumin n = 13, DMSO n = 14, HI n = 14), females (L, curcumin n = 14, DMSO n = 15, HI n = 17) and combined (males + females) (M, curcumin n = 27, DMSO n = 29, HI n = 31). (*p < 0.05). Abbreviations: CTX, cerebral cortex; EC – external capsule; HIP – hippocampus; PYR, pyriform cortex; STR, striatum; THAL, thalamus.

Intraperitoneal injection of curcumin (200 μg/g BW) in P7 mice immediately post-HI decreases phosphorylated STAT3 Y705 and S727 in hippocampus, while increasing ipsilateral PHB protein levels. (A) Western Blots for pSTAT3 (Y705) in ipsilateral and contralateral hippocampus from animals with HI, DMSO or 200 μg/g curcumin treatment and 1 h recovery. β-actin protein levels served as control. Note the increase of STAT3 Y705 ipsi- and contralaterally in the DMSO-treated group (19 and 16%, respectively) when compared to HI. Curcumin treatment decreases STAT3 Y705 protein levels ipsi- and contralaterally by 3 and 22%, respectively when compared to HI, and by 15 and 33% (p = 0.04) when compared to DMSO treatment. (B) Western Blots for pSTAT3 (S727) in ipsilateral and contralateral hippocampus from animals with HI, DMSO or 200 μg/g curcumin treatment and 1 h recovery. β-actin protein levels served as control. Note the increase of STAT3 S727 ipsi- and contralaterally in the DMSO-treated group (29 and 2%, respectively) when compared to HI. Curcumin treatment decreases STAT3 Y705 protein levels ipsi- and contralaterally by 3 and 10%, respectively when compared to HI, and by 25% (p = 0.009) and 12% when compared to DMSO treatment. (C) Western Blots for PHB in ipsilateral and contralateral hippocampus from animals with HI, DMSO or 200 μg/g curcumin treatment and 1 h recovery. β-actin protein levels served as control. Note the ipsilateral 13 and 19% (p = 0.0026) increase of PHB protein levels in the curcumin treated animals compared to HI and DMSO, respectively. Interestingly curcumin treatment results in contralateral 23 and 12% decrease of PHB protein levels compared to HI and DMSO littermates, respectively. PHB levels in DMSO-treated littermates were reduced by 4% in the ipsilateral and by 13% in the contralateral side compared to littermate HI animals. The ratio between pSTAT3 or PHB and β-actin immunoreactivity is shown in arbitrary units (AU) set as 100% for the first line of curcumin treated animals on each of the blots. “R” represents relative densitometry compared to β-actin, which was used as the internal loading control.

Cytotoxic effect of curcumin and formulated curcumin on other HCC cell lines (Huh7, Mahlavu, and PLC5). After separate treatment with 1, 3, 10, 30, 60, and 90 µM of conventional and formulated curcumin for 72 h, cell viability was determined by MTT assay and expressed as a percentage relative to the control group. Formulated curcumin was more effective than conventional curcumin on the basis of cell viability in the three cell lines tested. Formulated curcumin has higher cytotoxicity in (A) Huh7, (B) Mahlavu, and (C) PLC5 compared to conventional curcumin. ^#p < 0.05; ^##p < 0.01; ^###p < 0.005 compared to the control group (formulated curcumin). *p < 0.05; **p < 0.01; ***p < 0.005 compared to the control group (conventional curcumin). MTT, 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.