HMOX1 x KEAP1 x Nrf2

Lycium barbarum, commonly known as Goji berry, has been traditionally utilized in herbal medicine to enhance vitality and address reproductive health disorders. Polysaccharides derived from its fruit (LBPs) represent key bioactive components, which have been scientifically documented to exert protective effects on male reproductive function and to alleviate oxidative stress.

This study aims to evaluate the therapeutic potential of LBP-4 against triptolide (TP)-induced male reproductive toxicity and to elucidate the underlying mechanisms, with a specific focus on oxidative stress, apoptosis, and the Nrf2/Keap1 signaling pathway.

Male mice were co-administered TP (120 μg/kg/day) and LBP-4 (20/40/60 mg/kg/day)to assess reproductive recovery. Evaluations included: Body and organ indices: Body weight, testicular, and epididymal weights. Sperm parameters: Sperm count, motility, and malformation rate via computer-assisted sperm analysis (CASA).

Testicular tissue integrity and spermatogenic cell organization using hematoxylin-eosin (HE) staining. Oxidative stress markers: SOD, MDA, and GSH levels measured by biochemical assays. Testes from mice group (control, TP, H-LBP-4) underwent UID-mRNA-seq. DEGs were analyzed via GO and KEGG enrichment. Molecular pathways: Nrf2/Keap1 pathway gene/protein expression (QPCR, Western blot), Bax/Bcl-2 ratio, and caspase-3 and autophagy markers.

Reproductive Recovery: LBP-4 administration dose-dependently ameliorated TP-induced damage, significantly improving body weight, testicular and epididymal indices, and sperm parameters (count, motility, and morphology). Histological Improvement: LBP-4 restored testicular architecture and spermatogenesis, significantly increased mature sperm numbers, and mitigated cellular disarray. Higher doses of LBP-4 further alleviated atrophy and enhanced tissue organization. Oxidative Stress Mitigation: LBP-4 normalized the TP-altered levels of SOD (a 1.67-fold increase), GSH (a 21.86 % elevation), and MDA (a 54.90 % reduction), demonstrating dose-dependent efficacy. RNA-seq analysis revealed significant differential gene expression among groups: CON vs TP (448 upregulated/551 downregulated genes), H-LBP-4-TP vs TP (856 upregulated/1775 downregulated genes), and H-LBP-4-CON vs CON (3583 upregulated/4087 downregulated genes). Differentially expressed genes (DEGs) were enriched in pathways related to oxidative stress-induced apoptosis regulation, spermatogenesis, and spinal cord development in the CON vs TP group. Upregulated genes were associated with ubiquitin proteolysis and oxidative phosphorylation, while downregulated genes were linked to apoptosis and stem cell pathways in the H-LBP-4 vs TP comparison. Apoptosis Suppression: LBP-4 significantly downregulated the Bax/Bcl-2 ratio (an 87.76 % reduction), inhibited caspase-3 activation (a 1.15-fold decrease). Nrf2/Keap1 Pathway Activation: LBP-4 enhanced Nrf2 nuclear translocation and and elevated expression levels of the downstream antioxidant enzymes HO-1 and NQO1.

LBP-4 counteracts TP-induced male reproductive toxicity by activating the Nrf2/Keap1 pathway to alleviate oxidative stress, suppressing apoptosis via Bax/Bcl-2 and caspase-3 modulation, and restoring spermatogenic function. These findings validate Lycium barbarum's traditional use in reproductive health and propose LBP-4 as a promising adjuvant to enhance TP's clinical safety in cancer and autoimmune therapies.

We aimed to identify and isolate the metabolically active compounds from Cornus officinalis (C. officinalis) that underlie the biological effects previously observed in our in vitro and in vivo studies. Our prior findings demonstrated that C. officinalis promoted activation of the Keap1/Nrf2 pathway in a pancreatic β-cell line and delayed the onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. To characterize the metabolically active compounds within C. officinalis, the concentrated extract was fractionated by column chromatography, and evaluated for their effect on β-cell metabolic activity and expression of Nrf2 targets such as heme oxygenase-1 (HO1) and superoxide dismutase 2 (SOD2). Highly concentrated compounds of interest within metabolically active fractions were isolated using high performance liquid chromatography (HPLC). Elucidation of a highly abundant compound within C. officinalis extract was identified by mass spectrometry and nuclear magnetic resonance (NMR) as 5-hydroxymethylfurfural (5-HMF) which was demonstrated by immunoblotting to significantly increase SOD2 expression. Quantitative proteomics was performed on 5-HMF treated murine non-obese diabetic (NOD) islets and revealed increased expression of Nrf2 and downstream targets such as SOD2, GSTA3, GSTA4 and GCLC. Our findings suggest that 5-HMF is a highly abundant and metabolically active compound within C. officinalis that stimulates partial activation of the Keap1/Nrf2 pathway.

Osteoporosis, a prevalent metabolic bone disease affecting millions worldwide. Although MS-275 has been reported to inhibit oxidative stress, its ability to protect osteoblasts from oxidative stress damage has yet to be clarified. This study investigated whether MS-275 can inhibit oxidative stress and promote osteogenesis by activating the miRNA-200a/Keap1/Nrf2 signaling pathway.

In vitro, MC3T3-E1 cells underwent induction with carbonyl cyanide 3-chlorophenylhydrazone, leading to the establishment of an oxidative stress model, investigating the underlying mechanism. In vivo, using a rat model of ovariectomized osteoporosis, evaluating the effects of MS-275.

In vitro, MS-275 treatment of oxidation-induced MC3T3-E1 cells resulted in up-regulation of osteoblast protein, increased expression of miRNA-200a, increased binding of miRNA-200a to Keap1 mRNA, decreased expression of Keap1 protein, and dissociation of Nrf2 from Keap1. The expressions of total Nrf2, nuclear Nrf2 and HO-1 were increased, mitochondrial function was enhanced, and oxidative damage was reduced. However, these effects were reversed after interference with miRNA-200a. In vivo,MS-275 effectively enhanced the microstructural features of distal femoral trabecular bone, increased the mineralization capacity of osteoblasts, and promoted bone formation.

MS-275 can reverse oxidative stress-induced cell damage, promote bone healing, and improve osteoporosis by activating the miRNA-200a/Keap1/Nrf2 pathway.