*2.2. Redoxomics*

Modulation of Hsp72, HO-1, Thioredoxin, Sirtuins and γ-GC Liase, in MD Patients after Coriolus Mushroom Supplementation

Oxidative stress plays a role in the pathogenesis of a wide variety of pathological states [35–37]. ROS can oxidize membrane lipids generating lipid hydroperoxides and many aldehydes such as HNE, luminescent by-products, and isoprostanes. HNE can accumulate in cells in relatively high concentrations and cause cell toxicity. Recent studies have also shown that in response to environmental changes and other stressful conditions promoting proteotoxicity [38–40], cells adaptively activate synthesis and accumulation of several members of stress proteins, primarily Hsp70 and HO-1. As reported in Figures 2 and 3, mushroom supplementation with *Coriolus* biomass preparation resulted in up-regulation of the inducible isoforms of both Hsp70 and heme oxygenase-1 (HO-1), in lymphocytes (Figures 2a and 3a), a finding observed also in plasma, (Figures 2b and 3b), as compared to untreated group of MD patients. A representative Western blot obtained probing tissue samples with an antibody specific for the inducible isoform of heat shock proteins 70 (Hsp72) or Heme oxygenase are shown in Figure 2c,d and Figure 3c,d, respectively. Western blot analysis of the Thioredoxin protein also revealed a significant increase in the group of patients treated with *Coriolus* compared to control group, in lymphocyte and plasma (Figure 4a,b). A representative blot of thioredoxin protein is reported in Figure 4c,d. Similar results were also obtained analyzing sirtuin-1 expression. As shown in Figure 5a, Sirtuin-1 immunoreactivity was higher in lymphocytes of a group of MD patients treated for 2 months with *Coriolus* than in the untreated MD group. Consistent with this, plasma sirtuin-1 levels were higher in MD patients supplemented with mushrooms, as compared to the MD group of patients alone (Figure 5b). Representative blots of sirtuin-1 protein are reported in Figure 5c,d, respectively. Another important redoxomic component of vitagene network is γ-GC liase, the rate-limiting enzyme for intracellular glutathione (GSH) synthesis. Notably, GSH concentration and γ-GC liase activity are declining with age in the central nervous system (CNS), a condition associated with increased oxidative

stress [41]. Here we report that lymphocyte γ-GC liase levels were higher in MD patients supplemented with mushrooms as compared to the MD group of patients alone (Figure 6a). A representative blot of γ-GC protein is reported in Figure 6b.

**Figure 2.** Heat shock protein 70 levels in lymphocytes and in plasma from MD patients. Samples from MD patients were assayed for heat shock protein 70 (Hsp70) by western blot as described in Materials and Methods. A representative immunoblot is shown in (**c**,**d**). β-actin has been used as loading control. The bar graphs (**a**,**b**) show the densitometric evaluation and values are expressed as mean ± SEM of independent analyses on 22 patients (MD plus *Coriolus* biomass) and, respectively, on 18 patients (MD alone), per group. \* *p* < 0.05 vs. MD alone. D.U., densitometric units.

**Figure 3.** Heme oxygenase-1 levels in lymphocytes and in plasma from MD patients. Samples from MD patients were assayed for heme oxygenase-1 (HO-1) by western blot as described in Materials and Methods. A representative immunoblot is shown. β-actin has been used as loading control (**c**,**d**). The bar graph shows the densitometric evaluation (**a**,**b**) and values are expressed as mean ± SEM of independent analyses on 22 patients (MD plus *Coriolus* biomass) and, respectively, on 18 patients (MD alone), per group. \* *p* < 0.05 vs. MD alone. D.U., densitometric units.

**Figure 4.** Thioredoxin levels in lymphocytes and in plasma from MD patients. Lymphocyte samples (**a**) and plasma samples (**b**) from MD patients were assayed for thioredoxin (Trx) by western blot as described in Materials and Methods. A representative immunoblot is shown (**c**,**d**). β-actin has been used as loading control. \* *p* < 0.05 vs. MD alone. D.U., densitometric units.

**Figure 5.** Levels of sirtuin-1 in lymphocytes (**a**) and plasma (**b**) from MD patients. Samples from MD patients were assayed for sirtuin-1 by Western blot as described in Materials and Methods. Representative immunoblots are shown in the same figure (**c**,**d**). β-actin has been used as loading control. The bar graph shows the densitometric evaluation and values are expressed as mean ± SEM of independent analyses on 22 patients (MD plus *Coriolus* biomass) and, respectively, on 18 patients (MD alone), per group. \* *p* < 0.05 vs. MD alone. D.U., densitometric units.

**Figure 6.** γ-GC liase levels in lymphocytes from MD patients. Plasma samples from MD patients were assayed for γ-GC liase by western blot as described in Materials and Methods. A representative immunoblot is shown (**b**). β-actin has been used as loading control. The bar graph shows the densitometric evaluation and values are expressed as mean ± SEM of independent analyses on 22 patients (MD plus *Coriolus* biomass) and, respectively, on 18 patients (MD alone), per group (**a**). \* *p* < 0.05 vs. MD alone. D.U., densitometric units.
