Study on the anti-inflammatory activity of cyclohexene ether terpenes in spider fragrance in macrophages
Inflammation is a normal protective response to stimuli, tissue damage, and infection, and is a necessary physiological process for maintaining immune system homeostasis and healing. During the inflammatory process, the immune system produces high levels of pro-inflammatory mediators that play an important role in host cell defense. However, uncontrolled or prolonged inflammatory reactions are important causes of bodily damage or chronic diseases such as cancer, rheumatoid arthritis, and vascular diseases. Monocytes and macrophages are important defense lines of innate immunity and a significant source of a range of pro-inflammatory cytokines. When macrophages are stimulated by pathogens or endogenous factors, they secrete various cytokines including tumor necrosis factor alpha (TNF-a), interleukins (IL-1, IL-6, and IL-12), as well as chemokines, prostaglandins, leukotrienes, and complement. All of these inflammatory mediators work together to enhance vascular permeability and recruit immune cells. Currently, commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) still have adverse reactions such as gastric injury, bronchospasm, kidney and heart damage. Therefore, it is of great significance to explore new natural anti-inflammatory drugs with low toxicity and side effects from traditional Chinese medicine.
Valeriana jatamansi Jones, a plant of the Valeriana L. genus in the family Verbenaceae, is mainly distributed in Yunnan, Guizhou, Sichuan and other regions of China. It is a traditional herb commonly used by many ethnic groups in China. Its roots and stems have multiple effects such as calming the nerves, regulating qi and relieving pain, reducing inflammation and diarrhea, dispelling wind and dampness, and can be used for the treatment of abdominal distension and pain, injuries from falls and blows, vomiting and diarrhea, rheumatism and pain, menstrual disorders, sores and boils. Research has found that its chemical components mainly include cyclohexene ether terpenes, flavonoids, volatile oils, etc. Pharmacological studies have shown that one of the main components, cyclohexene ether terpenes, has antibacterial, antioxidant, antiviral and other effects. In our preliminary research, we found that the iridoid compounds isolated from spider fragrance have certain anti influenza virus activity. In order to further search for the active substances, we conducted a systematic chemical study on the site, tested the anti-inflammatory activity of 14 cyclohexene ether terpenes isolated from it, and further investigated the relevant mechanisms of action of compounds with significant anti-inflammatory activity.

Macrophages play a crucial role in immune and inflammatory responses. When activated by internal and external stimuli, they induce the activation of cellular signaling pathways such as MAPKs and NF kB, thereby regulating the expression of various inflammation related genes downstream, promoting cascade cascade cascade reactions, and releasing a large amount of inflammatory factors for defense. Excessive immune activation often causes damage to the body, ultimately leading to further deterioration of the disease.

INOS and COX-2 are both inducible enzymes that are not expressed or expressed at low levels in most tissues, but can show high expression under the action of some inflammatory cytokines. INOS is widely involved in the expression of inflammatory cytokines and the production of reactive nitrogen and oxidative products, playing a key role in the occurrence and development of inflammatory pathology. COX-2 is a key enzyme that catalyzes the conversion of arachidonic acid into prostaglandins, and therefore plays an important role in the induction and maintenance of inflammation. The expression of both factors and the production of inflammatory cytokines TNF-a and IL-1 are regulated by transcription factors NF kB and STAT3.

In the inactive state, NF kB and the inhibitory protein IkBa exist as a complex in the cytoplasm. Many extracellular ligands (such as TNF-a, IL-1, LPS, etc.) can quickly trigger the classic NF kB signaling pathway and further activate downstream kinases, causing phosphorylation and subsequent ubiquitination degradation of IkBa, free NF kB translocation to the human nucleus, activating a series of pro-inflammatory target genes, promoting the expression of inflammatory factors and iNOS, COX-2. STAT3 plays an important role in regulating cell growth, proliferation, differentiation, and apoptosis, and can be activated by various cytokines and growth factors, such as IL-6 and EGF/EGFR. Its two key phosphorylation sites, tyrosine 705 and serine 727, are regulated by multiple pathways including mTOR, MAPKs, and PKC. MTOR, as an important regulatory factor involved in various life processes including protein and lipid synthesis, autophagy, glycolysis, and inflammation, can significantly activate STAT3 through phosphorylation. Activated STAT3 further induces the expression of downstream inflammatory genes, releases IL-6, C3, etc., and further feedback stimulates the STAT3 signaling pathway, forming a sustained inflammatory microenvironment, which is an important cause of inflammation persistence and cytokine storm.

In addition, inflammasomes, as a type of cytoplasmic protein complex, are an important component of the human natural immune system and play a crucial role in the occurrence and development of various inflammation related diseases. Among them, NLRP3 inflammasome is currently the most widely studied type of inflammasome. The uncontrolled activation of inflammasome NLRP3 can promote the excessive secretion of inflammatory cytokine IL-1 by activating caspase-1, which is one of the important causes of inflammatory body damage.

Therefore, the above mediators and signaling pathways are considered drug targets for the prevention and treatment of inflammatory diseases, and there is a close relationship and synergistic effect between them. This study screened the anti-inflammatory activities of 14 cyclohexene ether terpenoids isolated from spider fragrance and found that compounds 1, 2, 3, 5, 8, and 9 had significant anti-inflammatory activities. We focused on the negative regulatory effects of cyclohexene ether terpenoids 1 and 2 in the macrophage inflammation model. The study found that compounds 1 and 2 significantly reduced the expression of inflammatory factors NO, IL-1, IL-6, and TNF-a induced by LPS, leading to a decrease in the expression levels of iNOS, COX-2, and NLRP3. Further experimental research results showed that compounds 1 and 2 inhibited LPS stimulated IkBa phosphorylation, effectively suppressing the activation of the NF kB signaling pathway. Meanwhile, the inhibition of mTOR and STAT3 phosphorylation by the compound further reduced the secretion of inflammatory mediators and their subsequent feedback effects.

In summary, compounds 1 and 2 have a negative regulatory effect on LPS induced macrophage inflammation by downregulating the NF kB and mTOR/STAT3 signaling pathways, inhibiting inflammasome formation, and significantly reducing the expression of iNOS and COX-2, thereby regulating the release of NO and various inflammatory factors, demonstrating good anti-inflammatory activity. This study indicates that compounds 1 and 2, represented by cyclohexene ether terpenes, in Spider Fragrance have significant anti-inflammatory activity and are likely the main active ingredients for Spider Fragrance’s good anti-inflammatory effects in traditional medicine. This study provides experimental basis and scientific theoretical basis for the comprehensive development and utilization of the anti-inflammatory effects of spider fragrance.