Effects of Gastrodin on Analgesia and Inhibition of Ferroptosis

Background: Gastrodin possesses low toxicity and a broad range of pharmacological activities and exhibits benecial effects in neurological diseases. This study investigated the effects of gastrodin (GAS) on analgesic, anti-inammatory, anxiolytic and inhibition of ferroptosis. Materials and Methods: The chronic inammatory pain model of C57BL/6J mice was established by hindpaw injection of complete Freund’s adjuvant (CFA). After GAS treatment, Thermal hyperalgesia test, Mechanical allodynia test, Elevated plus-maze (EPMT) and Open-eld test (OFT) were performed to assess the behavioral changes of pain and anxiety. mRNAs of FTHI, GPX4, HO-1 and PTGS2 were measured by RT-qPCR. Results: In CFA-injected C57BL/6 mice, we found that the mechanical and thermal pain threshold was increased with treatment of GAS. In EPMT, the number of entries in open arms and retention times of open arms were increased by GAS. In the OFT, the time spent in the central area was also increased. Furthermore, GAS enhanced mRNA expressions of FTHI, GPX4 and H0-1, as well as decreased the expression of PTGS2 in a dose-dependent manner. Conclusion: GAS is effective in the treatment of mice chronic inammatory pain and anxiety-like behaviors. It maybe exhibit potential neuroprotective effects through inhibition of ferroptosis.


Background
Ferroptosis is a unique iron-dependent form of regulated cell death (1). The accumulation of lipid peroxidation products and lethal reactive oxygen species (ROS) is the main characteristic of ferroptosis (2). Ferroptosis, as a way to promote cell death, may be implicated in the occurrence and development of many diseases. Studies have shown the importance of ferroptosis in many diseases of the central nervous system, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS), Traumatic brain injury (TBI) (3).
Gastrodiae Rhizoma (Tianma), a notable Chinese herb, is dry tubers of Gastrodia elata Blume which belongs to Orchidaceae. Gastrodiae Rhizoma is considered a top-grade medicine described to treat the hypertension of liver-yang hyperactivity in the tradition Chinese medicine. Studies have shown that the application of gastrodia elatahas biological activities of anticonvulsion, antioxidation, neuroprotection, anti-denguevirus, anti-cardio-cerebral-vascular diseases, anti-in ammation (4). The major active component and material basis of Gastrodia elata is Gastrodin (GAS). GAS, a chemical compound that known as 4-hydroxybenzyl alcohol-4O-β-D-glucopyranoside, is isolated from the rhizome of Gastrodia elata. Furthermore, the molecular formula of GAS is C 13 H 18 O 7 , and its chemical structural formula is shown in Fig. 1. GAS has numerous pharmacological activities including analgesic(5), antidepressant(6), anxiolytic(7), anti-in ammatory(8), antiobesity (9) and memory and retrieval improvements (10,11).
Among them, analgesic, antioxidant, anti-in ammatory and neuroprotective effects are the main research hotspots in recent years. Recent ndings suggest that GAS exerts a protective effect on primary neural progenitor cells (NPCs) by resisting amyloidβ (Aβ) (1-42)-induced neurotoxicity (12). In the meantime, GAS increased the expression of HO-1, Nrf2 and GPX4 protein in Rat Glioma Cell Line C6, which protected Rat Glioma Cell Line C6 from ferroptosis induced by H 2 O 2 (13). In recent years, several neuroprotective mechanisms of GAS have been fund. However, the study regarding to the effects of Gas on ferroptosis is rare. GAS was also reported to display powerful anti-in ammation properties. Based on the above research progress and analysis, it is speculated that GAS might be a potential therapeutic for the inhibition of ferroptosis. This study was designed to explore the effects of GAS on analgesic, antiin ammatory and anxiolytic. We also examined whether GAS can exhibit neuroprotective effect through inhibition of ferroptosis.

Animals and grouping
Male C57BL/6J mice (aged 8 weeks, weighing 21-25g) were purchased from Chengdu Dashuo Laboratory Animal. Animals were housed in groups of six mice with a temperature (20 ± 2℃), humidity (55 ± 15%) and lighting (12 h light/dark cycle, lights on at 7:00 AM). All animals must adapt to conditions for at least 7 days after they arrived. Food and water were freely available.
Experimental designs and GAS treatment 10ul CFA (50%) was injected intraplantar subcutaneously into the left hindpaws of mice to established chronic peripheral in ammatory pain. In the control group, the same volume of SAL was injected into the hindpaws of mice. GAS was dissolved in saline before use. The mice were intraperitoneally injected with GAS (100, 200 mg/kg) after CFA insult GAS or saline was used repeatedly in mice once a day for 2 weeks.

Mechanical allodynia
Mechanical allodynia was assessed with a set of von Frey laments on day 1,4,7, and 14. Mice were placed on a wire mesh covered with organic glass and acclimated to the environment at least 30 minutes prior to test. Start with 0.4 mN(#2.44) lament and stimulate the center of left hindpaw until lament bending for 3s, and the mice have reactions like licking foot or foot lifting.

Thermal hyperalgesia
After 14 days of administration, the temperature of the hot plate was set to 55℃. The left hindpaw of mice was placed on the hot plate, and time was recorded when the mice had reactions like foot lifting.  (Table 1).

Results
Effects of GAS on CFA-induced mechanical and thermal hypersensitivity After CFA was injected into mice, mechanical thresholds were determined on day 1, 4, 7, and 14. As shown in Fig.2, on the rst day after CFA injection, the mechanical pain threshold of the model group was signi cantly lower than blank group, and the left hindpaw of mice was obviously swollen, indicating that the chronic in ammatory pain model was successfully established. The paw withdrawal threshold of CFA-injected mice signi cantly decreased after CFA injection for 1-4 days. Meanwhile, the administration of GAS (100 and 200 mg/kg) increased the paw withdrawal threshold in CFA-injected mice. GAS also attenuated thermal hyperalgesia in CFA-injected mice (Fig.3). Moreover, GAS dose-dependently increased the mechanical and thermal pain threshold in mice.

Effects of GAS on CFA-induced anxiety-like behavior
Anxiety the GAS-treated group were increased (Fig.4A, B). In the OFT, compared with the blank group, the time spent in the central area decreased in the model group. while the GAS (100 and 200 mg/kg) reversed the reduction caused by CFA (Fig.5). The results show that GAS attenuated CFA-induced anxiety-like behavior.
mRNA expression changes GAS on chronic in ammatory pain model The mRNA expressions of FTHI, GPX4, HO-1 and PTGS2 in the ACC and L4-5 of experimental mice on day 14 after CFA injection were detected through Real-time Quantitative PCR. The relative expression levels of each group of genes were shown in Fig 6(A, B).
In the ACC and the spinalcord of the rat lumbosacral enlargement, both FTH1 and GPX4 were signi cantly decreased on the model group as compared with blank group. Meanwhile, we found that CFA elevated the expressions of PTGS2 and HO-1. Finally, compared with the model group, FTH1, GPX4 and HO-1 in GAS groups were signi cantly increased while PTGS2 decreased in a dose-dependent pattern. Take together, GAS improved FTHI, GPX4, HO-1 and PTGS2 mRNA expressions, and decreased the production of ROS.

Discussion
Acute in ammatory pain induced by injection of CFA. In this process, rats were allergic to mechanical allodynia and thermal hyperalgesia, and the pain-induced anxiogenic effect lasted for more than 14 days (14). Clinically, it has been reported that chronic pain leads to mental problems such as anxiety and depression, which seriously reduces the quality of life of patients and hinders their normal life (15). GAS is a phenolic glucoside with signi cant analgesic and anti-in ammatory effects. In the CFA-induced chronic in ammatory pain model, we found that mechanical and thermal pain threshold was increased with treatment of GAS in a dose dependent pattern. In addition, the number of entries in open arms and retention times of open arms were increased by GAS. These studies further con rmed that GAS has powerful analgesic, anti-in ammatory and anti-anxiety effects in the chronic in ammatory pain model of mice. GAS exerted analgesic and anti-in ammatory effects by decreasing the activation of astrocyte and microglia and the induction of TNF-α and IL-6 in the ACC(16). In a mouse model of chemotherapeutic agent-induced neuropathic pain, 5-HT 1A receptor can mediate the powerful antinociceptive of GAS(5).
In ammatory disease (ID)is a series of diseases characterized by in ammatory response, and ferroptosis is closely related to in ammatory response (17). There are some in ammatory factors related to the metabolism of peroxides and arachidonic acid in ferroptosis tissues(18). Studies have shown that both ferroptosis and in ammatory diseases have the depletion of Gx4 and GSH, the increase of lipid peroxidation products, and the interruption of iron metabolism (19). At present, although a variety of molecular mechanisms and signaling pathways can lead to ferroptosis, Iron metabolism and lipid peroxidation signaling are the main way to regulate ferroptosis (20). During iron metabolism, Excessive iron leads to ferroptosis by producing ROS. Ferritin heavy chain 1(FTH1), as an iron storage protein complex, is involved in the uptake of excessive iron (2). we found that GAS increased the expression of FTH1, and thus balanced intracellular iron levels. The heme oxygenase-1 (HO-1), a major intracellular source of iron (21), plays an important role in ferroptosis and in ammation. It was reported that p38 MAPK phosphorylation could mediate the protective effect of GAS on H 2 O 2 -induced oxidative stress (22).
GAS could ameliorate MPP+-induced oxidative stress by regulating the expression of HO-1 in human dopaminergic cells (23). We also demonstrated that GAS increases HO-1 expression, which accelerates the decomposition of heme and inhibits in ammation. In addition, the expressions of glutathione peroxidase4 (GPX4) and prostaglandin-endoperoxide synthase2 (PTGS2) are also important for the induction of ferroptosis.
In our experiments, GAS signi cantly upregulates the expression of FTH1and GPX4, decreases PTGS2 expression, suggests that GAS against ferroptosis by reducing lipid peroxidation. CFA-induced chronic in ammatory pain is accompanied by the ferroptosis of neuronal cells, and GAS has an inhibitory effect on ferroptosis, which is one of the possible mechanisms to protect neuronal cells. In order to make the argument more su cient, in the future, it is necessary to further study the mechanism of GAS inhabiting ferroptosis at cellular levels.

Conclusion
In conclusion, our research shows that GAS has obvious analgesic and anti-anxiety effects on CFAinduced chronic in ammatory pain in mice. At the same time, GAS maybe exhibit potential neuroprotective effects through inhibition of ferroptosis. The data from the present study provide a theoretical basis for GAS in the treatment of neurological diseases, and promote in-depth research and application of GAS.
Declarations Figure 1 The chemical structural formula of GAS. Effect of GAS on results of OFT (*P<0.05 compared with blank group)