Total glucosides of paeony inhibit NLRP3/caspase-1/GSDMD-mediated inflammation and pyroptosis in C3H/HeJ alopecia areata mice

One of the most prominent causes of alopecia areata (AA) is chronic inflammation of the hair follicles. Inhibiting cellular pyroptosis, a form of inflammatory programmed cell death, is crucial for reducing follicular inflammation in the skin. Total glucosides of paeony (TGP) possess anti-inflammatory properties across a broad range of illnesses. However, the role of TGP in AA and its relationship to pyroptosis remains unclear. A chronic unpredictable mild stress (CUMS) approach was used to create an AA mouse model. TGP suspension and MCC950 were administered to AA mice via gavage. HE staining, ELISA, immunohistochemistry, immunofluorescence, RT-qPCR, and Western blotting were performed to detect pathological changes in the skin and to investigate the levels of inflammatory factors and pyroptosis-related proteins, as well as the potential mechanisms of TGP's effects. TGP reduced hair loss, increased the number of hair follicles in skin tissues, and decreased inflammatory markers (IL-6, TNF-α, IL-18, and IL-1β) in AA mice. MCC950 significantly reduced the levels of NLRP3/caspase-1/GSDMD-mediated pyroptosis-related proteins (NLRP3, ASC, caspase-1 p10, and GSDMD-N), as well as inflammatory factors. TGP markedly inhibited NLRP3/caspase-1/GSDMD-mediated cellular pyroptosis in a concentration-dependent manner. TGP suppresses the NLRP3/caspase-1/GSDMD signaling cascade in the skin tissues of AA mice, thereby reducing cellular pyroptosis and inflammation. TGP may be a potential therapeutic agent for AA.


INTRODUCTION
Alopecia areata (AA) is an autoimmune alopecia areata disease without scarring characterized by a chronic inflammatory response to the destruction of the immune privilege of the hair follicle [1], with a global prevalence of 2%.It can occur at any age, most commonly in individuals who are young and middle-aged, and there are no significant gender differences [2].Furthermore, AA is prone to recurrence and increases the risk of other autoimmune illnesses such hypothyroidism, systemic lupus erythematosus, vitiligo, and psoriasis [3,4].The clinical diagnosis of AA is made using dermoscopy and histopathological examination [2], based on the typical clinical manifestations of patients with patchy alopecia of the peripheral scalp, or even total hair loss in severe cases [5], with inflammatory cells spread into the hair follicle epithelium and the periphery of the skin tissues [6].AA has a significant impact on the patient's quality of life and may cause psychological disorders such as anxiety and depression [7,8].There are no treatments available to prevent or cure AA, and current therapeutic agents focus on inhibiting or modulating the invasion of hair follicles by inflammatory cells, and include externally applied classes (clobetasol propionate, minoxidil, anthralin, and diphenylcyclopropenone), topically injected classes (triamcinolone acetonide and etanercept), phototherapy (psoralen plus ultraviolet A (PUVA)), and oral classes (JAK inhibitors and cyclosporine A).Although these drugs are effective, they are not recommended for prolonged use because of the possibility of relapse and obvious side effects [9].The search for new drugs with high efficacy and low side effects for the treatment of AA remains worth exploring.
Total glucosides of paeony (TGP) is a collection of active ingredients extracted from the root of Paeonia lactiflora that has few side effects and regulate immunity, inhibit inflammation, and protect the liver [10,11].Since 1998, the State Food and Drug Administration of China has approved TGP for the treatment of rheumatoid arthritis [12].TGP has been shown to inhibit lymphocyte infiltration and the activation of NLRP3 inflammasomes in order to alleviate dry skin syndrome [13]; it can also regulate the balance of pro-inflammatory/anti-inflammatory cytokine (IL-2/IL-7) secretion to inhibit atopic dermatitis [14].TGP also has good clinical efficacy in skin immune diseases like psoriasis [15], systemic lupus erythematosus [16], psoriasis [17] and lichen planus [18].As a result, we hypothesized that TGP may have some therapeutic effects on AA; however, there are fewer such studies, and the mechanism of action of TGP in AA remains unknown.
TGP has been shown to suppress pyroptosis, hence reducing inflammatory response and promoting tissue healing [19,20].Unlike apoptosis and necrosis, pyroptosis is a genetically controlled inflammatory cell death that is primarily caused by the activation of inflammasomes and the activation of inflammatory caspase, and excessive pyroptosis causes a variety of autoinflammatory and autoimmune disorders [21,22].Inflammatory vesicles can activate caspase family proteins, which control both classical and non-classical cellular pyroptosis processes [23].Caspase-1 plays a role in the classical cellular pyroptosis process.Inflammatory vesicles like NLRP3, NLRC4, IPAF, and AIM2 stimulate and cleave pro-caspase-1 to generate active caspase-1, which subsequently cleaves the substrate Gasdermin D (GSDMD) or inflammatory factor precursors (e.g.pro-IL-1β and pro-IL-18), leading to cell expansion until the cell membrane ruptures, releasing inflammatory factors and triggering pyroptosis [24][25][26][27][28]. Previous research has revealed that pyroptosis is a crucial factor in the autoimmune process [29][30][31].TGP may help with AA symptoms by reducing pyroptosis.NLRP3/caspase-1/GSDMD is known to be a critical pathway that initiates pyroptosis [32,33], but no study has investigated its regulatory mechanism in AA or if TGP improves cutaneous alopecia via this pathway.Based on this, we investigated the effects of the NLRP3/caspase-1/GSDMD pathway on pyroptosis and inflammatory responses in AA mice skin tissues, as well as the potential mechanisms by which TGP modifies inflammatory responses in AA.The study intends to bring new insights into the treatment of autoimmune illnesses using Chinese medicine.

Lab animals
Henan Kangda Laboratory Animal Co. (Zhengzhou, China) provided us thirty-six healthy female C3H/HeJ mice aged 6-8 weeks.Before the experiment, the mice were acclimatized to the following conditions: keeping 12 hours of light/dark cycle, a room temperature of 25℃ and a relative humidity of 50%, and free access to food and drink.inhibitor group (MCC950, 20 mg/kg).Both TGP and MCC950 were administered via gavage and given once a day at noon.Except for the control group, each set of six mice underwent a 21-day CUMS stress experiment with seven different stimuli on seven days per week, with the stress selection and occurrence randomized.The mice were executed at the completion of the stress experiment (the 22nd day).The seven stimuli were: circadian reversal, water and food fasting for 24 hours, noise stimulation for 3 hours, tilted cage overnight for 8 hours, swim for 3 minutes in icy water at 4℃, tail suspension for 1 minute, and horizontal electric shock at 100 Hz for 15 minutes.

Skin tissue sample preparation
Mice were anesthetized with 1% pentobarbital sodium (40 mg/kg) intraperitoneally, and blood was collected by removing the eyes, catching the blood in anticoagulant-free EP tubes, and leaving to stand at 37℃ for 1 hour.The blood samples was then centrifuged at 4℃ for 10 minutes at 3,000 r/min using a refrigerated tabletop centrifuge, and the supernatant (blood serum) was transferred to EP tubes before being frozen in the -80℃ refrigerator.
The skin on the back of euthanized mice was depilated with depilatory cream and separated into sections.Some were preserved in 4% paraformaldehyde solution, while others were frozen at -80℃ in the refrigerator.

HE-staining
Fixed skin tissues were taken from 4% paraformaldehyde solution, dehydrated, embedded, and cut into 4 μm tissue sections using a microtome (RM2235, Leica Germany).Tissue sections were dewaxed with xylene and hydrated using an alcohol gradient.After 15 minutes of staining with hematoxylin (C0107, Beyotime, Shanghai, China), the sections were differentiated with 1% acidic alcohol (containing 70% hydrochloric acid) for 30 seconds and washed with running water before immersing in 0.5% eosin (G1100, Solarbio, Beijing, China) for 3 minutes.The sections were again treated using alcohol gradient dehydration and xylene clear treatment.Finally, the slices were sealed with neutral gum (G8590 Solarbio).The number of skin follicles and inflammatory infiltration were observed under the microscope.

ELISA test
The plate was incubated at 37℃ with 50 μL of analytical buffer and 50 μL of sample for 2 hours.Biotinylated antibody (100 μL) was added to the plate and incubated for 1 hour at room temperature.The plate was placed to the plate and treated for an hour at 25℃ with light protection.Then, 100 μL of TMB chromogenic reagent was put in and treated for 20 minutes with light protection.Finally, 50 μL of termination buffer was added to stop the reaction.The samples' absorbance was determined at 450 nm using microplate reader, and the content was estimated.
Microscopic examination and photos were taken.

Data-based Analysis
Statistical analyses and graphs were created with GraphPad Prism 9.0 (GraphPad Inc., La Jolla, CA, USA).SPSS 26.0 software (SPSS Inc., Chicago, IL, USA) was used to examine data for significance.The data were presented as mean ± standard deviation.
ANOVA, t-test, and chi-squared were used to compare groups.All experiments had significant statistic findings (*P<0.05).

Ethical statement
This study was approved by Huiji District People's Hospital of Zhengzhou Ethics Committee.

TGP attenuates histopathologic skin damage in AA mice
We randomly chose one mouse from each group of C3H/HeJ mice to compare hair shedding on the mice's backs under various treatment circumstances.In comparison to the control group, mice in the AA model group showed significant hair loss over a large area of the back, and the skin appeared to be exposed with AA characteristics; following gavage of different concentrations of TGP to treat AA mice, the hair loss on the back improved, as did the range of exposed skin and the hair regeneration rate and hair thickness in mice: TGP-L>TGP-M>TGP-H (Figure1A-1C).The results of HE staining of skin tissue sections, as shown in Figure 1D, showed that sebaceous glands (yellow arrows) and hair follicles (green arrows) were significantly reduced in the AA model group, and a large number of inflammatory cells, primarily neutrophils and lymphocytes (blue arrows), infiltrated around them.In contrast, the control group had essentially no or few inflammatory cells, and an abundance of hair follicles.
Compared to the AA group, the TGP-L group demonstrated a growth in the amount of sebaceous glands and a reduction in the infiltration of inflammatory cells in skin tissues; the TGP-M group showed an increase in hair follicles in skin tissues, with hair growing to the site of sebaceous glands and encapsulated by inner hair root sheaths, and a reduction in the amount of inflammation-related cells; and the TGP-H group showed a significant decrease in the number of inflammatory cells in skin tissues, and had abundant sebaceous glands and hair follicles.It was established that TGP gavage stimulated the production of hair follicles in the skin tissues and reduced damage from inflammation in the skin tissues of AA mice, and the impact was dose dependent.

TGP lowers serum inflammatory factor levels in AA mice
The pathogenesis of AA is followed by the release of a number of inflammatory substances by inflammatory cells (e.g., neutrophils, lymphocytes), which enhances the immune system's response and accelerates the development of autoimmune illnesses [6].We used ELISA to measure the amounts of inflammatory factors IL-6, TNF-α, IL-18, and IL-1β in the serum of different mice groups.Figures 2A-2D shows that the AA group had significantly higher levels of all four inflammatory factors in their serum in contrast to the control group.However, the levels of IL-6, TNF-α, IL-18, and IL-1β in the serum gradually decreased with increasing TGP treatment concentration.
The current investigation found that TGP gavage successfully reduced inflammatory components in mice's serum.

TGP lowers inflammatory factor levels in skin tissues of AA mice
We then measured the expression levels of inflammatory factors in the skin tissues of mice in each group.The mRNA expression of IL-6, TNF-α, IL-18, and IL-1β in skin tissues as measured by RT-qPCR were consistent with that of serum, all of them were significantly high in the AA group, and decreased continuously with the increase in the concentration of TGP treatment (Figures.3A-3D).Western blot analysis revealed that the AA group had significantly higher amounts of IL-6, TNF-α, IL-18, and IL-1β compared to the control group.However, as the concentration of TGP treatments increased, the levels of these inflammatory factors decreased (Figure 3E-3I).TGP was also shown to diminish the levels of inflammatory elements in the skin tissues of AA mice, demonstrating that it greatly inhibited the inflammatory response in AA animals in vivo.

NLRP3/caspase-1/GSDMD-mediated pyroptosis in AA mice skin tissues
Pyroptosis is a pro-inflammatory kind of programmed cell death that can be initiated by inflammasome vesicles, resulting in robust inflammatory and immunological responses [35].TGP has been shown to effectively decrease the inflammatory response in AA mice, hence it is thought that it does so by reducing pyroptosis signaling.NLRP3 inflammatory vesicles trigger the release of IL-1β, IL-18, and the development of GSDMD pores by activating caspase-1, which facilitates pyroptosis [36].As a result, the current study, we designed to give AA mice the abdomen of the

MCC950 attenuates inflammatory response in AA mice
After gavage of MCC950, we investigated the influence of the NLRP3/caspase-1/GSDMD pathway on inflammation in AA mice.The exposed area of skin on the back of mice after MCC950 injection was significantly reduced compared to the AA group (Figure 5A), and the hair regeneration rate and hair thickness were significantly up-regulated in mice (Figures 5B-5C).HE staining showed that the hair follicles in the AA group were significantly reduced and a large number of inflammatory cells infiltration was seen in the surrounding area; however, the amount of hair follicles was increased and the number of inflammatory cells was significantly reduced in the MCC950 group (Figure 5D).MCC950 gavage treatment dramatically reduced the expression of inflammatory factors (IL-6, TNF-α, IL-18, and IL-1β) in AA mice skin tissues, as shown by Western blot analysis (Figure 5E-5G).
This study found that MCC950 gavage was able to reduce the response of inflammatory in AA mice, resulting in improved hair loss, revealing that the NLRP3/caspase-1/GSDMD pathway facilitated pyroptosis, which in turn enhanced the inflammatory response in AA mice.In summary, we predicted that TGP reduced inflammation in AA mice by decreasing NLRP3/caspase-1/GSDMD-mediated pyroptosis.

AA mice
To put the theory to the test, we looked at how TGP affected pyroptosis-related proteins, as well as the number of caspase-1 p10 and Tunel-positive cells in mice skin tissues.Western blot analysis revealed that TGP gavage significantly inhibited the expression of NLRP3, ASC, caspase-1, GSDMD and GSDMD-N in AA mice skin tissues, with the inhibitory effect concentration-dependent (Figure 6A-6E); immunohistochemical results also demonstrated that TGP was able to inhibit NLRP3 and caspase-1 p10 level in AA mice skin tissues in a concentration-dependent manner (Figure 6F).Furthermore, when TGP gavage concentration increased, the number of caspase-1 p10 and Tunel-positive cells in AA mice's skin tissues reduced (Figure 6G-6H).The preceding investigations indicated that TGP reduced NLRP3/caspase-1/GSDMD-mediated pyroptosis in the skin tissues of AA mice, which may be an essential mechanism for its inflammation-suppressive impact.

DISCUSSION
AA is a genetic autoimmune illness that occurs when immune cells penetrate the hair follicle during the anagen phase, when the hair follicle's immune system is impaired [37].During the hair cycle, the epithelium of the hair follicle maintains a zone of relative immunological immunity, which promotes regular hair development [2].An autoimmune reaction can disrupt the hair cycle and prematurely terminate growth, followed by follicular atrophy and atrophic alopecia [1].Treatment for AA relies on decreasing autoimmunity by inhibiting the release of inflammatory factors and immune cell infiltration.C3H/HeJ mice, which develop spontaneous alopecia due to autoimmune processes, are widely regarded as the best animal model for researching AA [38].As a result, we chose C3H/HeJ mice to develop an AA model via CUMS induction.TGP, as an anti-inflammatory herbal component, can alleviate skin tissue pathology by controlling lymphocytes, reducing inflammation, and inhibiting the secretion of inflammatory molecules [39].Our experimental results showed that as the dose of TGP administration increased, skin hair loss on the back of AA mice continued to improve and the invasion of inflammatory cells in the skin tissues gradually decreased.The expression levels of IL-6, TNF-α, IL-18, and IL-1β in the serum and skin tissues of the mice gradually decreased, which was in line with the reported.Furthermore, the CUMS method is frequently used to create depression models in mice, which cause significant psychological stress, resulting in loss of pleasure, weight loss, elevated corticosterone levels, thymic atrophy, and adrenal hypertrophy [40,41].TGP can also reduce neuroinflammation and provide antidepressant effects [42].As a result, TGP can also reduce the psychological stress caused by CUMS in mice.
During the development of AA, lymphocytes such as CD8 + T and CD4 + T infiltrated hair follicles rapidly.CD8 + T cells target the inner section of the hair follicle, while CD4 + T and NK cells concentrate near the outer root sheath of the hair follicle, and these lymphocytes release pro-inflammatory cytokines such as TNF-ɑ and IL-6, resulting in a breakdown in the follicle's immunological response [43,44]

CONCLUSION
This research describes a new potential therapy method for AA in which TGP reduces the inflammatory response of AA by modifying NLRP3/caspase-       G-H: Immunofluorescence results showed that TGP was capable of dose-dependently lower the intensity of caspase-1 p10 and Tunel fluorescence, as well as the number of NLRP3 inhibitor MCC950 gavage treatment and to demonstrate that lesion development in AA mice occurs as a result of pyroptosis by observing changes in AA mice's pemphigus and detecting the levels of NLRP3/caspase-1/GSDMD-associated factors in AA mice organisms.After Western blot analysis, the pyroptosis-related protein proteins (NLRP3, ASC, caspase-1, and GSDMD-N) were significantly elevated in the skin tissues of mice in the AA group in contrast to the control group (Figure4A-4C); immunohistochemistry also found that the number of staining for NLRP3 and caspase-1 p10 was significantly increased in the sections of the AA group in contrast to the control group (Figure4D-4E), demonstrating that the NLRP3/caspase-1/GSDMD pathway was activated in AA mice.Double staining of sections for caspase-1 p10 and Tunel revealed that the number of caspase-1 p10 and Tunel-positive cells was significantly higher in the sections of the AA group compared to the control group (Figure4F-4G), implying that the NLRP3/caspase-1/GSDMD pathway mediated pyroptosis in the skin tissues of AA mice.We gavaged the NLRP3 inhibitor MCC950 intraperitoneally into AA mice and found a significant decrease in the level of the pyroptosis-associated proteins NLRP3, ASC, caspase-1, and GSDMD (Figure4A-4E), as well as a significant decrease in the number of caspase-1 p10-and Tunel-positive cells (Figure4F-4G), demonstrating that MCC950 can effectively inhibit NLRP3/caspase-1/GSDMD-mediated pyroptosis, indicating that the pathological development of pyroptosis signaling.
. The conventional mechanism for pyroptosis is NLRP3/caspase-1/GSDMD.The formation of NLRP3 inflammasomes, which are made up of the innate immune receptor protein NLRP3, the articulin ASC, and the inflammatory protease caspase-1, is triggered by cellular stimulation.This leads to the activation of caspase-1, and the formation of the cleavage protein GSDMD is accompanied by the production of numerous inflammatory factors, including L-1β, IL-18, and other inflammatory factors [45].Cell membrane pores, swelling, and rupture are then caused by the release of IL-18 and IL-1β into the extracellular space, which further triggers a cascade of amplified inflammatory response.This can then lead to the release of additional inflammatory factors, like TNF-α and IL-6, and ultimately completes the pyroptosis program [46-49].T lymphocytes can induce cellular pyroptosis via the NLRP3 inflammatory programmed death pathway[50], whereas activation of NLRP3 inflammasome cells promotes the development of AA in C3H/HeJ mice [51].Both NK and CD 8+ T cells have been shown to kill Leishmania protozoa-infected cells, resulting in the release of DAMPs, activation of NLRP3 inflammatory vesicles, and IL-1β, which exacerbate skin ulceration [52].These findings indicate that both T cells and NK cells located within the hair follicle or in the root sheath region outside the hair follicle can activate the NLRP3/caspase-1/GSDMD pathway, hastening the development of AA.Furthermore, NLRP3/caspase-1/GSDMD pathway-mediated pyroptosis causes psychiatric illnesses like as depression [53], increasing the risk of AA treatment.Therefore, inhibiting NLRP3/caspase-1/GSDMD is extremely beneficial for the therapy of AA.We found that MCC950 effectively suppressed the expression of pyroptosis-related proteins (NLRP3, ASC, caspase-1 p10, and GSDMD) and inflammatory factors (TNF-α, IL-6, L-1β, and IL-18) in the skin tissues of AA mice.TGP, which had a similar effect to MCC950, also inhibited the expression of pyroptosis-related proteins and inflammatory factors in a concentration-dependent manner.Related proteins and inflammatory factors are concentration-dependent.The number of caspase-1 p10 and Tunel-positive cells in AA mice's epidermal tissues decreased considerably following TGP treatment.These results indicate that TGP can substantially reduce inflammation in AA mice by reducing NLRP3/caspase-1/GSDMD-mediated pyroptosis.TGP may be a candidate herbal ingredient for the treatment of AA, which opens up possibilities for the creation of new AA medications.Furthermore, AA pathogenesis is caused by a complex interaction of genes and factors.Future research into other potential methods of TGP regulating AA, as well as toxicity evaluation and clinical trials, is required to promote the development of TGP Chinese medicinal preparations and their practical application in the clinic.

Figure 1 .
Figure 1.TGP inhibited histopathologic skin deterioration in AA mice.A: Representative image of skin alopecia on the back of mice.B-C: Changes in hair regeneration rate and hair thickness in mice.D: HE staining results showed that compared to the control group, the number of hair follicles in the AA group was significantly reduced and a large number of inflammatory cell infiltration could be seen in the surrounding area.Skin tissue damage was gradually improved with the administration of TGP-L, TGP-M, TGP-H.(20×, bar=100 μm).n=6, *P<0.05.

Figure 3 .
Figure 3. TGP lowered inflammatory factor levels in skin tissues of AA mice.A-D: TGP inhibited mRNA levels of IL-6, TNF-α, IL-18, and IL-1β in skin tissues in a dose-dependent manner, measured by qRT-PCR experiment.E-I: TGP inhibited IL-6, TNF-α, IL-18, and IL-1β protein levels in skin tissues in a dose-dependent manner, as demonstrated by Western blot analysis.n=6, *P<0.05.

Figure 5 .
Figure 5. MCC950 attenuated inflammatory response in AA mice.A: Representative photos of dorsal skin alopecia in mice.B-C: Changes in hair regeneration rate and hair thickness in mice.D: HE staining of skin tissue demonstrated that gavage with MCC950 dramatically improved skin tissue injury.(20×,bar=100 μm).E-G: Western blot analysis revealed that the MCC950 group had considerably lower levels of inflammatory markers in skin tissue than the AA group.n=6，*P<0.05.
The experiments followed the Guidelines for the Management and Use of Laboratory Animals established by Huiji District People's Hospital of Zhengzhou Ethics Committee.(No.KZ-20220509).

Table 1 . Arrangements of the seven stimuli that C3H/HeJ mice experienced over 21 days
disease [54], it is necessary to increase publicity efforts to promote relevant knowledge to AA patients and the general public, as well as to guide the public toward a civilized and healthy lifestyle.
line with market standards.Furthermore, given that psychological stress and lifestyle habits (e.g., smoking, drinking, diet, and sleep) are important factors in triggering or exacerbating AA