KEAP1 x NLRP3 x Nrf2

The root of Saposhnikovia divaricata has been traditionally used in Chinese medicine to treat inflammation and immune related skin diseases, such as skin allergy and itchy rash. However, scientific research supporting its efficacy, particularly in curing inflammation from acne, is still scarce.

To evaluate the therapeutic effects of essential oil of Saposhnikovia divaricata (EOSD) on Cutibacterium acnes (C. acnes)-induced acne and investigate its mechanisms.

An acute acne model was established by injecting C. acnes into mouse ears, followed by topical EOSD treatment (0.25 %, 0.5 % and 1 %). Ear tissues were analyzed 24 h post-treatment. In vitro, C. acnes-stimulated J774A.1 macrophages were treated with EOSD (3, 10 and 30 μg/ml) to assess mitochondrial damage, oxidative stress, and inflammatory responses. Key markers were analyzed using transmission electron microscopy, real-time PCR, immunoblotting, ELISA, and immunofluorescence. Nrf2 knockdown was achieved via siRNA transfection.

In vivo, C. acnes suppressed Nrf2 activation, increased ROS levels, and triggered NLRP3 inflammasome activation, leading to inflammation. EOSD restored Nrf2 activity, reduced ROS levels, and inhibited NLRP3 activation. In vitro, EOSD disrupted Nrf2/Keap1 interaction, enhanced Nrf2 nuclear translocation, reduced mitochondrial damage, and suppressed pyroptosis by downregulating IL-1β, Caspase-1, GSDMD, and NF-κB signaling. These effects were reversed by Nrf2 knockdown.

EOSD alleviates oxidative stress and modulates NLRP3 inflammasome activation through upregulating Nrf2 pathway and inhibiting NF-κB pathway, as well as improving mitochondrial homeostasis and inflammatory responses in acne. These findings suggest that EOSD may be a promising therapeutic agent for the treatment of acne vulgaris.

Fever is a prevalent clinical symptom and is usually caused by inflammation or infection. Persistent high fever can lead to delirium, coma and convulsions, causing brain damage. Angong Niuhuang Pill (ANP), a traditional Chinese emergency medicine, has been employed in clinical practice for centuries, with well-documented antipyretic effects. However, the molecular mechanisms underlying its temperature-regulating effects remain to be fully elucidated.

This research aimed to analyze the effective components of ANP and examine its therapeutic effects and probable mechanisms on fever.

In the present study, Chemical constituents of ANP and its blood-absorbed components were identified using UHPLC-Q-Exactive-Orbitrap-MS. Network pharmacology research was employed to investigate the multi-target, multi-pathway mechanisms of ANP therapy for fever. The antipyretic activity of ANP was evaluated using the LPS-induced fever model in rats. The peripheral blood leukocyte counts of the rats were detected using routine blood tests. The levels of TNF-α and IL-6 in serum and the levels of COX-2 and PGE2 in the hypothalamus were measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, the transcriptome sequencing of rat hypothalamus tissue was utilized to explore the mechanism of antipyretic action at the gene level. Simultaneous immunofluorescence and Western Blot (WB) techniques were used to confirm the expression of various mRNAs and proteins in the hypothalamus, including COX-2, NLRP3, NRF2, TLR4, KEAP1, p-P65, P65, TNF-α and IL-17A. Then, the LPS-Induced RAW264.7 cell model was used to explore the regulatory role of ANP on macrophage polarization phenotype. Protein and mRNA expression levels of iNOS, CD86, and CD206 in RAW264.7 macrophages were evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and WB analysis.

A total of 169 chemical components were identified in ANP, 13 of which were detected in serum. Network pharmacology research indicated that 13 components in ANP interacted with 91 critical targets to produce their effects. In vivo experiments demonstrated that ANP treatment effectively relieved elevated body temperature and reduced the inflammatory response in rats. The ELISA results showed that all dose groups of ANP could significantly down-regulate the levels of TNF-α and IL-6 in serum, as well as the levels of COX-2 and PGE2 in the hypothalamus. The results of transcriptome sequencing and Western Blot showed that ANP could up-regulate the expression of NRF2 and KEAP1 protein, and down-regulate the expression of TLR4, IL-17A, p-P65/P65, TNF-α, and NLRP3 protein. Meanwhile, ANP significantly inhibited the LPS-induced overproduction of NO, IL-6, and TNF-α inflammatory factors in RAW264.7 cells. Furthermore, ANP suppressed the mRNA and protein expression of iNOS and CD86, enhanced NRF2 protein expression, and reduced p-P65/P65 expression.

This work analyzed the intricate chemical constituents of ANP and demonstrated that ANP can efficiently exert antipyretic and anti-inflammatory effects by inhibiting the TLR4/NF-κB/NLRP3 inflammasome axis and activating the NRF2/KEAP1 signaling pathway. The study provided some guidance for subsequent clinical applications and research on ANP.

Intervertebral disc degeneration (IDD) is a key contributor to lumbar degenerative diseases and chronic low back pain. Accumulating evidence indicates that Sequestosome 1 (SQSTM1/p62), a multifunctional adaptor protein, plays a pivotal role in IDD pathogenesis through its regulation of autophagy, oxidative stress, inflammation, and programmed cell death. This review summarizes the multifaceted functions of SQSTM1 in the context of IDD, including its involvement in the autophagy-lysosome pathway, antioxidant defense via the Keap1-Nrf2 axis, activation of the NF-κB signaling and NLRP3 inflammasome, and modulation of apoptosis, pyroptosis, and ferroptosis. Moreover, SQSTM1 contributes to extracellular matrix degradation by upregulating matrix metalloproteinases and downregulating their inhibitors. Given its dynamic expression during disc degeneration, SQSTM1 holds promise as both a biomarker for IDD progression and a therapeutic target. Potential strategies targeting SQSTM1 include the use of autophagy inducers, inflammatory pathway inhibitors, and ferroptosis/pyroptosis modulators. However, challenges remain in precisely modulating SQSTM1 activity and translating findings into clinical therapies. Future research leveraging advanced technologies such as single-cell RNA sequencing, proteomics, and organoid models is essential to unravel the complex, stage- and cell-specific roles of SQSTM1 in IDD. Understanding these mechanisms may open new avenues for effective treatment and improved patient outcomes in degenerative spinal disorders.