Interleukin-35 Ameliorates Cyclophosphamide-induced Ovarian Injury by Inhibiting PANoptosis.

Abstract

Introduction: Cyclophosphamide (CTX) is widely used in chemotherapy; however, its clinical application is frequently accompanied by gonadotoxic effects, which can result in impaired ovarian function and subsequent infertility in women. Consequently, effective strategies to mitigate chemotherapy-induced ovarian injury remain an important unmet need. Interleukin-35 (IL-35), a recently identified member of the IL-12 cytokine family, has been implicated in immune regulation and the maintenance of immune homeostasis. In parallel, accumulating evidence indicates that ovarian damage involves multiple forms of regulated cell death rather than a single apoptotic pathway. PANoptosis, an integrated cell-death program encompassing pyroptosis, necroptosis, and apoptosis, has recently emerged as a key mechanism in tissue injury. To date, whether IL-35 participates in CTX-induced ovarian injury and whether it modulates PANoptosisrelated pathways has not been clearly elucidated.

Materials and methods: Female C57BL/6 mice were randomly divided into four groups: control, CTX treatment, IL-35 treatment, and combined CTX + IL-35 treatment. Histopathological alterations in ovarian tissue were assessed using hematoxylin and eosin staining, whereas ELISA, qRT-PCR, immunohistochemistry, and western blot assays were subsequently conducted to investigate potential molecular mechanisms.

Results: IL-35 showed a protective effect against CTX-induced ovarian injury. Serum FSH levels were reduced. Serum E2 and AMH levels were increased. Ovarian histopathological structure was improved. CTX treatment increased oxidative stress markers. These markers included ROS, SOD, and MDA. CTX treatment increased apoptosis markers (Bax and Bcl2). CTX treatment increased pyroptosis markers (NLRP3 and IL-1β). CTX treatment increased necroptosis markers (p-MLK and p-RIPK1). IL-35 pretreatment reduced all listed molecular changes. Further analysis showed enhanced activation of the ovarian Nrf2/HO-1 signaling pathway after IL-35 pretreatment during CTX exposure.

Discussion: IL-35 preserves ovarian reserve during CTX challenge by activating Nrf2/HO-1 to restore redox balance, thereby coordinately inhibiting pyroptosis, necroptosis, and apoptosis-key drivers of chemotherapy-induced follicular depletion.

Conclusion: IL-35 reduced CTX-induced ovarian injury. This effect occurs through the regulation of oxidative stress and PANoptosis. These findings indicate the therapeutic potential of IL- 35 for ovarian injury caused by CTX.