Salidroside Protects Bovine Mammary Cells From Inflammation by Activating Mitophagy

Salidroside, a natural compound extracted from the roots and rhizomes of Rhodiola plants, has emerged as a strikingly effective protector of bovine mammary epithelial cells under inflammatory attack, according to a new study published in the journal Animal Diseases. The research, conducted entirely in cell culture using the MAC-T bovine mammary epithelial cell line, shows that pretreatment with salidroside dramatically reduces the production of inflammatory signaling molecules triggered by lipopolysaccharide, the major endotoxin of Escherichia coli and a central driver of the mammary gland inflammation known as mastitis. The work identifies a specific molecular circuit through which the compound operates, linking cellular energy sensing to the disposal of damaged mitochondria, and it offers a mechanistic foundation for developing plant-derived alternatives to the antibiotics on which mastitis control has long depended.

The stakes are considerable for the global dairy industry. The study’s authors cite previous estimates that roughly 60 to 70 percent of lactating cows are affected by mastitis, with clinical cases accounting for 21 to 23 percent of total incidence and economic losses estimated at 15 to 45 billion US dollars. Lipopolysaccharide, the primary pathogenic component of E. coli, initiates the inflammatory cascade in bovine mastitis, and bovine mammary epithelial cells sit at the front line of that assault. These cells perform a dual role, synthesizing and secreting the components of milk while simultaneously serving as the primary cellular barrier against pathogen invasion. When lipopolysaccharide strikes, the resulting inflammatory injury is characterized not only by extensive activation of proinflammatory signaling pathways but also by profound mitochondrial dysfunction, a combination that the new study places at the center of its investigative strategy.

To build their experimental model, the researchers first established safe and effective doses. Neither lipopolysaccharide at concentrations up to 200 micrograms per milliliter nor salidroside at concentrations up to 1,000 micromolar induced significant cytotoxicity in MAC-T cells over a 12-hour treatment period, as measured by a CCK-8 viability assay. When the cells were exposed to a range of lipopolysaccharide concentrations, the secretion of the inflammatory cytokines TNF-alpha and IL-6 rose significantly at 5 micrograms per milliliter, with higher concentrations producing fluctuating rather than steadily escalating responses. The team therefore selected 5 micrograms per milliliter as the inflammatory stimulus. Screening salidroside pretreatments across the same 12-hour window revealed pronounced anti-inflammatory activity between 200 and 500 micromolar, and the lowest effective dose of 200 micromolar was chosen for all subsequent experiments to avoid unnecessary high-dose exposure while still significantly inhibiting the lipopolysaccharide-induced secretion of TNF-alpha, IL-6, and IL-1beta.

With the model established, the researchers documented the protective effect of salidroside in detail. Lipopolysaccharide stimulation significantly increased the messenger RNA levels of four proinflammatory cytokines, TNF-alpha, IL-6, IL-1beta, and IL-18, while simultaneously suppressing the messenger RNA levels of the autophagy-related genes ATG5, ATG12, and ATG16L1. At the protein level, lipopolysaccharide decreased the ratio of LC3B-II to LC3B-I, reduced the abundance of the autophagy proteins Beclin-1, PINK1, and Parkin, and increased levels of p62, a substrate that accumulates when autophagic degradation falters. Salidroside intervention reversed all of these detrimental changes. To visualize autophagic flux directly, the team employed a tandem mRFP-GFP-LC3 reporter system, in which yellow puncta mark autophagosomes and free red puncta, generated when the green fluorescent signal is quenched in acidic lysosomes, mark autolysosomes. Lipopolysaccharide treatment substantially reduced both puncta populations, indicating impaired flux, whereas salidroside restored both, providing direct visual evidence that the compound reestablishes functional autophagic degradation.

The mitochondrial dimension of the injury proved equally central. Lipopolysaccharide stimulation significantly depleted intracellular ATP, dissipated the mitochondrial membrane potential as measured by the red-to-green fluorescence ratio in JC-1 staining, and triggered abnormal opening of the mitochondrial permeability transition pore, detected through a reduction in calcein-AM fluorescence intensity. Each of these defects reflects a stage in the collapse of mitochondrial integrity that the authors describe as a pivotal event driving the exacerbation of inflammation. Depolarized mitochondria leak reactive oxygen species that act as endogenous danger signals, prompting the dissociation of thioredoxin-interacting protein and its binding to NLRP3, which drives assembly of the NLRP3 inflammasome, activation of caspase-1, and the maturation and release of the proinflammatory mediators IL-1beta and IL-18. Salidroside pretreatment significantly mitigated the ATP depletion, restored the membrane potential, and suppressed the abnormal pore opening, positioning the compound as an upstream interceptor of the mitochondrial collapse that fuels the inflammatory cascade.

The mechanistic core of the study lies in the AMPK/mTOR signaling axis, a central upstream pathway governing cellular energy sensing and mitochondrial quality control. Compared with cells receiving lipopolysaccharide alone, salidroside-treated cells showed significantly increased ratios of phosphorylated to total AMPK and correspondingly decreased ratios of phosphorylated to total mTOR, indicating activation of the energy sensor and suppression of its downstream target. To test whether this axis is genuinely required for the protective effect rather than merely correlated with it, the researchers used compound C, a specific pharmacological inhibitor of AMPK. Blocking AMPK activity markedly reversed salidroside’s suppression of proinflammatory cytokine messenger RNA levels, and it also blunted the compound’s restoration of autophagy-related gene expression, the LC3B-II/I ratio, Beclin-1 abundance, and p62 degradation. The increase in yellow and red puncta in the flux assay was likewise largely abrogated by the inhibitor, demonstrating that the AMPK/mTOR axis is a critical upstream regulator of the autophagic response that salidroside induces.

Parallel inhibitor experiments confirmed the necessity of mitophagy itself. When cells were pretreated with 3-methyladenine, an inhibitor of autophagy initiation, the cytoprotective effect of salidroside was markedly abrogated and cytokine messenger RNA levels rose significantly. Cyclosporin A, used to block mitophagy, reversed the salidroside-induced upregulation of ATG5, ATG12, and ATG16L1, suppressed the increases in the LC3B-II/I ratio and Beclin-1, raised p62 levels, and critically attenuated the upregulation of PINK1 and Parkin, effectively blocking the initiation of the mitophagy program. Under these conditions the anti-inflammatory protection conferred by salidroside substantially decreased, with cytokine levels rising significantly. Compound C similarly compromised the mitochondrial benefits, reducing intracellular ATP content, reversing the restoration of membrane potential, and permitting abnormal reopening of the permeability transition pore. Together these findings establish a coherent causal chain in which salidroside activates AMPK, suppresses mTOR, drives PINK1/Parkin-mediated clearance of damaged mitochondria, preserves mitochondrial energy metabolism and membrane integrity, and thereby limits the downstream inflammatory cascade.

The authors are careful to delineate the boundaries of their evidence. All experiments were conducted in vitro using the MAC-T cell line, which cannot fully recapitulate the complex physiological microenvironment, tissue architecture, and immune cell recruitment within the living bovine mammary gland, so the protective efficacy and molecular mechanisms observed here may not entirely reflect clinical scenarios. The core findings have not yet been validated in primary bovine mammary epithelial cells, a verification the researchers identify as necessary future work. Translational questions also remain open: although dietary supplementation would offer operational convenience for herd-scale interventions, orally ingested salidroside may undergo ruminal microbial metabolism that compromises its postruminal absorption and bioavailability. The authors point to rumen-protected formulations as one possible solution and to drug-loaded hydrogels delivered by intramammary infusion as another, though the latter would require further evaluation of biocompatibility and formulation costs. Future in vivo studies, they write, must systematically evaluate pharmacokinetics, tissue distribution, biosafety, and milk residue profiles across delivery strategies.

The study also acknowledges that salidroside’s anti-inflammatory effects may not be governed exclusively by the AMPK/mTOR to PINK1/Parkin axis. Nrf2-mediated antioxidant defenses are intimately linked to mitochondrial redox homeostasis and can modulate mitochondrial injury and mitophagy, while the canonical inflammatory pathway NF-kappaB is closely tied to mitochondria-derived oxidative stress, and potential cross-talk among these systems remains to be systematically investigated. Within those limits, the findings add salidroside to a growing list of phenylethanoid glycosides, including forsythoside A, that appear to share a mechanism of cytoprotection through targeted activation of PINK1/Parkin-mediated mitophagy. For a disease that costs the dairy sector billions annually and whose management has been constrained by antibiotic resistance and drug residues in milk, a plant-derived compound that intercepts mitochondrial dysfunction upstream of the inflammatory burst represents an intriguing candidate, one whose real-world value now awaits the verdict of live-animal trials.

Subject of Research: A cell-culture study of salidroside's protective effects against LPS-induced inflammatory injury in bovine mammary epithelial cells via AMPK/mTOR-regulated mitophagy

Article Title: Salidroside alleviates LPS-induced inflammatory injury in MAC-T cells by promoting mitophagy and improving mitochondrial function via the AMPK/mTOR pathway

Article References: Huang, Q., Cao, H., Geng, Y., Peng, Z., Lu, X., Zhang, S., Zu, N., Cao, L., Zheng, X., & Qi, Z. (2026). Salidroside alleviates LPS-induced inflammatory injury in MAC-T cells by promoting mitophagy and improving mitochondrial function via the AMPK/mTOR pathway. Animal Diseases, 6(1), Article 62. https://doi.org/10.1186/s44149-026-00283-1

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00283-1

Keywords: salidroside, bovine mastitis, MAC-T cells, mitophagy, PINK1, Parkin, AMPK/mTOR, lipopolysaccharide, mitochondrial dysfunction, inflammation, dairy cattle, mammary epithelial cells