Bo Wen,Cong Ma,Qin Zhang*,Yang Shi,Mi-Feng Liu,Xiao-Meng Huo,Wen Gu,Pei-Pei Shao,Ping Tang

1Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,Beijing 100010,China; 2Beijing Jingmei Group General Hospital,Beijing 102300,China.

Abstract Background:The study was conducted with the interest of exploring the effects of Qingre Yangyin Chushi pill as anti-inflammatory agent in gout treatment based on the NLRP3/GSDMD coke death pathway.Methods:In this study,48 male SD rats were randomly divided into 6 groups,namely model group,blank control group (BC),Qingre Yangyin Chushi pill low-dose treatment group (MSD1),medium-dose treatment group (MSD2),high-dose treatment group (MSD3),and colchicine group (PC),with eight members in each group.The BC and model groups were administered with saline twice a day.The MSD1,MSD2,and MSD3 groups were administered with Qingre Yangyin Chushi pill at a dose of 1.8 g·kg?1,3.6 g·kg?1,and 7.2 g·kg?1 twice a day.The colchicine group was administered with a colchicine suspension at a dose of 0.6 × 10?3 g kg?1 twice a day for 7 days.After gavage of animals in each group for 4 days,the rats' ankle joints were injected with sodium urate suspension × 3 days to induce a gouty arthritis model.Then,serum and tissue samples were collected on the third and seventh day after gavage.The synovial tissue from the rat ankle joints was taken,and immunohistochemical technique was used to detect NOD-like receptor protein 3 (NLRP3),inflammatory bodies,cysteine protease-1 (Caspase-1),and Gasdermin-D protein (GSDMD) expression.Image-Pro Plus image analysis system was used to measure the average integrated absorbance and calculate IHS.Integral enzyme-linked immunosorbent assay was used to determine the interleukin 1β (IL-1β) and interleukin 18 (IL-18) expression of tumor necrosis factor-α (TNF-α).Western blot was used to detect NLRP3,Caspase-1,GSDMD,and apoptosis-associated speck-like protein containing a CARD (ASC) expression levels.Results:After 48 hours of modeling,compared with the PC group,the arthritis indices of the MSD1,MSD2,and MSD3 groups were insignificant (P > 0.05).The levels of IL-1β,IL-18,and TNF-α were lower in the PC and MSD2 groups compared with the BC group.However,compared with the control group,MSD1,MSD2,and PC groups had lower levels of IL-1β and IL-18.Regarding TNF-α levels (P< 0.05); compared with the PC group,the levels of IL-1β,IL-18,and TNF-α in MSD2 decreased more significantly(P < 0.05).The levels of NLRP3,Caspase-1,GSDMD,ASC,IHS score,and mRNA were lower in the PC and MSD2 groups (P < 0.05) compared with the control group.The MSD1 and MSD2 groups had lower levels of NLRP3,Caspase-1,GSDMD,ASC protein levels,IHS points,and mRNA levels (P <0.05) compared with the PC group.Moreover,NLRP3,Caspase-1,GSDMD,ASC protein levels,IHS points,and mRNA levels were more reduced in the MSD2 group (P < 0.05).Conclusion:Qingre Yangyin Chushi pill can inhibit the activation of the NLPR3 inflammatory complex,reduce the production of GSDMD protein,regulate the occurrence of pyroptosis,reduce the expression of inflammatory factors,and thus reduce arthritis.All these processes are achieved through the NLRP3/GSDMD pathway.

Keywords:QingRe YangYin ChuShi pill,Gout,Inflammation,GSDMD,Pyroptosis,Gouty arthritis

Background

Gout is a metabolic disease associated with excessive circulating uric acid,which causes monosodium urate crystal to deposit on tissues [1].Acute Gouty Arthritis(AGA) is a severe inflammatory response that begins in the synovium and causes monosodium urate (MSU)crystals to deposit on joints and surrounding tissues.As an acute rheumatic disease,AGA’s primary clinical characteristics are inflammatory cell infiltration,swollen joints,and severe pain.The main histological characteristics of this process are proliferation of synovial lining cells and infiltration of neutrophils,monocytes or macrophages,and lymphocytes [2].The pathogenesis of gout has been deeply examined.MSU crystal activates the NLRP3 inflammasome and results in the secretion of IL-1β,the activation of neutrophils,the release of proinflammatory cytokines and inflammatory chemokines such as TNF-α and IL-6,and the aggregation of inflammatory cells at the site to trigger an inflammatory cascade reaction.On the other hand,MSU can directly activate the NLRP3 inflammasome by inducing reactive oxygen species,promoting the maturation of pro-caspase-1,cleaving GSDMD protein,and activating IL-1β and IL-18,which leads to the pyroptosis of mediation cells [3].

The Qingre Yangyin Chushi pill is a hospital preparation developed by prescription to clear away heat,nourish Yin,and remove dampness.It was created by Professor Wang Weilan,a late famous traditional Chinese medicine research doctor.The pill has been in clinical use for more than 10 years for treating various rheumatic diseases in active stages,and it has achieved good clinical effects [4–7].The Qingre Yangyin Chushi pill is composed of honeysuckle,forsythia,scutellaria barbata,polygonum cuspidatum,cortex dictamni,glabrous greenbrier rhizome,white peony root,dried and prepared rehmannia root,cassia twig,and radix aconiti.Previous studies suggest that the Qingre Yangyin Chushi pill increases the pain threshold of mice and inhibits inflammatory ear swelling and foot swelling in mice,which may be related to the reduction of inflammatory mediators [8,9].Based on this,we used the improved Coderre method to establish an animal model of gout and observed interventions of the Qingre Yangyin Chushi pill on the NLRP3/GSDMD pathway and effects on related inflammatory cytokines.We also explored possible mechanisms to provide an experimental foundation for the clinical application of this drug.

Materials and methods

Animals

Fifty male Sprague Dawley rats,weighing 180–220 g each,aged 6–8 weeks,with certificate number 11400700382137,were purchased from Beijing Charles River(ethical approval number:IRM-DWLL-2019048).

Main drugs and reagents

Drugs and reagents used were:sodium urate (Sigma,batch number BCBW1849); Tween 80 (Beijing Solarbio,batch number 822L0419); Qingre Yangyin Chushi pill (Beijing Hospital of Traditional Chinese Medicine,Capital Medical University,batch number 1811035); colchicine (KPC Pharmaceuticals,Inc,batch number 180705-01); TIANScript RT KIT,SuperReal PreMix Plus (SYBR Green); TRIzol,Tiangen Biotech (Beijing) Co.,Ltd.(Cat.No.KR104-02); TRIzol,Tiangen Biotech (Beijing) Co.,Ltd.(Cat.No.FP205); Invitrogen (Cat.No.15596-026); BCA protein assay kit (German Edeka Zentrale,batch number PP0102); Anti-NLRP3 antibody (American Ambion Company,ab214185);β-Actin antibody (Tianjin Sungene Biotech Co.,Ltd.,batch number KM9001); Anti-ASCC antibody(American Santa Cruz Biotechnology,SC-514414);Anti-Caspase-1 antibody (American Ambion Company,ab1872); and Anti-GSDMD antibody (American Ambion,ab219800).

Main instruments

The main instruments used included the following:paw volume meter (YLS-7C,Jinan Yiyan Technology Development Co.,Ltd.); hand-held homogenizer(Model No.:F6/10,Technology Department of Shanghai Jingxin Experimental Equipment); vortex mixer (QL-902,Haimen Kylin-Bell Lab Instruments Co.,Ltd.); centrifuge (Centrifuge 5415D,Eppendorf);biological spectrophotometer(BioPhotometer,Eppendorf); fluorescence quantitative PCR instrument(Connect CFXTM,BIO-RAD); high-speed centrifuge(Eppendorf,Germany); transference decoloring shaker(TS-8,Haimen Kylin-Bell Lab Instruments Co.,Ltd.);electrophoresis apparatus (Beijing Liuyi Instrument Factory); refrigerated centrifuge (5418R,Eppendorf,Germany); vertical electrophoresis tank (DYCZ-20C,Beijing Liuyi Instrument Factory); transfer tank(DYCP-40C,Beijing Liuyi Instrument Factory);microscope (BX51T-PHD-J11,Japan Olympus);CMOS (Japan Olympus); multifunctional true-color cell image analysis management system (Image-Pro Plus,Media Cybernetics,USA); microtome (RM2015,Leica,Germany);centrifuge(Eppendorf-5430,Eppendorf,Germany); cryogenic refrigerator (Model MDF-382E,Sanyo,Japan); pipette (Eppendorf,Germany); and thermostatic water bath (Model DSHZ-300,Jiangsu Taicang Medical Instruments Factory).

Animal experiment

Establishment of rat model.Rats were anesthetized by ether.Using a 6-gauge injection needle inserted into the articular cavity in the direction of 30–40° along the inner Achilles tendon,50 μl of normal saline was injected into the right ankle of the rats in the blank comparison group (BC),and 50 μl of sodium urate suspension (with a concentration of 25 g·l-1) was injected into the articular cavity of the rats in the other groups.The bulge on the opposite side of the joint capsule was used as the injection criterion.Next,a small amount of erythromycin ointment was applied to the injection site of the articular cavity to prevent infections.Normal saline and sodium urate solution were injected continuously for 3 days.After all rat models were established,the treated rats had significantly higher gait and joint swelling indexes than those in the BC group,indicating successful modeling.

Preparation of sodium urate solution.Sodium urate crystal (1250 mg) was added into 45 ml of normal saline,following which 5 ml of Tween 80 was added.The mixture was heated up and stirred well to prepare 50 ml of sodium urate solution.A Tachypiens Amebocyte Lysate (TAL) test was used to detect the presence of endotoxin.

Observed indicators and evaluation methods.Fifty rats were randomized into the BC group; model group;low-dose group (MSD1),medium-dose group (MSD2),and high-dose group (MSD3) of the Qingre Yangyin Chushi pill; and the colchicine group (PC),with 8 in each group.Rats in the BC and model groups were intragastrically administered 3 ml of normal saline twice a day.Rats in the MSD1,MSD2,and MSD3 groups were administered 1.8 g·kg?1,3.6 g·kg?1,and 7.2 g·kg?1,respectively,of the suspensions of the Qingre Yangyin Chushi pill (with concentrations of 0.06 g·ml?1,0.12 g·ml?1,and 0.24 g·ml?1,respectively),3 ml each time,twice a day,for 7 consecutive days.Rats in the colchicine group were administered 6 ×10?4g·kg?1colchicine suspension(with a concentration of 0.02 g·l?1),6 ml each time,twice a day,for 7 consecutive days.During this period,rats in each group were given access to food and water ad libitum.Models were established after animals in each group were administered intragastrically for 1 h on day 5.At 6 h,12 h,24 h,and 48 h after modeling,the ankle circumference of each rat was measured to calculate the joint swelling index.Other indicators were observed after modeling for 3 days.The joint swelling index was defined as [(joint circumference at the time of measurement-initial circumference)/initial circumference].The initial circumference was the circumference at 0 h.

Collection of samples

Serum and tissue samples were collected on day 7 after intragastric administration,that is,at 1 h after modeling on day 3.The rats were anesthetized with ether.Under aseptic conditions,3 ml of venous blood was drawn from the abdominal cavity,placed at room temperature for 20 min,and centrifuged for 10 min in a centrifuge at 3000 r·min?1.The serum was collected according to the instructions of the ELISA kit.After the blood was taken,the rats were sacrificed by cervical dislocation.At 0.5 cm in front of and at the back of the right posterior ankle,each rat was cut and skinned.On an ice plate,synovial tissues were isolated from the ankle quickly and carefully,and packaged.One part was placed in 10% formaldehyde solution for more than 24 hours to be fixed for immuno-histochemistry (IHC),while the other part was packaged into cryotubes and stored in a refrigerator at ?80 °C for Western blot analysis.

Real-time PCR detection of mRNA expression of genes related to NLRP3/GSDMD pathway

After the stored synovial tissues of the ankle were taken out,TRIzol was used to extract the total RNA in the tissue sample,and the integrity of 5μl of extracted RNA was examined by 1% agarose gel electrophoresis.The TIANScript RT KIT was used for reverse transcription,and the test was performed according to the product instructions.β-actin was used as the reference gene,and the relative quantitative analysis of genes was made on CFX Manager software according to the 2-ΔΔCtalgorithm.The primers were synthesized by Sangon Biotech (Shanghai) Co.,Ltd.The specific series are shown in Table 1.

Table 1 PCR primer sequences

Detection of expression of proteins related to the NLRP3/GSDMD pathway by Westernblot

Synovial tissue was cut into small pieces using scissors,and 200–400 μl of lysis buffer was added for each 20 mg of tissue.The tissue was homogenized with a hand-held electric homogenizer for about 1 min or until it was fully lysed.After being centrifuged at 12,000 rpm for 10 min,the supernatant was taken to determine the protein content in each sample by Coomassie blue staining (Bradford method).Please refer to the product instruction for specific steps.Ten percent (10%) separation gel and 4% stacking gel were prepared.According to the protein assay result,the corresponding volume of total protein samples and 5 ×buffer gel electrophoresis was added for sample loading,electrophoresis,and film transferring.The transferred film was put in 5% skimmed milk powder,sealed up for 1 h at room temperature under slow shaking on the shaker,and incubated overnight at 4°C with antibodies ASC (1 :300),Caspase-1 (1 :300),NLRP3 (1 :800),GSDMD (1 :800),and β-actin (1 :1000).It was then washed with 1 × TBST 3 times,10 min each time,added into the secondary antibody (1 :3000),shaken slowly for 60 min at room temperature in a dark place,and washed with 1 × TBST 3 times,10 min each time.After exposure and film processing,the gray value was analyzed using ImageJ software.

Detection of expression of related pathway proteins by IHC

The paraffin-embedded tissue section of ankle prepared with the conventional method was taken,deparaffinated,and placed in citrate buffer (pH 6.0) for 10 min for antigen retrieval.Next,the section was washed with phosphate buffer solution (PBS) 3 times,5 min each time,combined with a 3% hydrogen peroxide solution,and placed at room temperature for 25 min.Then,it was combined dropwise with goat serum to react for 20 min and then with ASC (1 :150),Caspase-1 (1 :200),NLRP3 (1 :500),GSDMD (1 :800),and β-actin (1 :1000) and left overnight at 4 ℃.Next,it was washed with PBS 3 times,5 min each time,and combined with a ready-to-use secondary antibody (SP-9000) from Zhongshan Goldenbridge Technology Co.,Ltd.for 20 min at 37 ℃,and once again washed with PBS 3 times,5 min each time.SA/HRP was added dropwise to the section at 37 ℃ to react for 20 min,followed by washing with PBS 3 times,5 min each time.After the DAB color developed,the section was washed thoroughly with water.The cell nucleuses underwent the hematoxylin counterstain for 1 min,and then were fully washed,differentiated with 1% acid alcohol,blued with 1% aqueous amine,fully washed again,and dehydrated as follows:70% ethanol for 5 min,80% ethanol for 5 min,90% ethanol for 5 min twice,95% ethanol for 5 min twice,and 100%ethanol for 5 min twice.Finally,the section was transparentized with xylene for 5 min twice and sealed with neutral resin.The section was observed by microscope,and the IHS score of each group was recorded.

IHS [10]=grade of positive cell number (A) × color intensity grade of positive cells (B).

A is the grade of the positive cell number:0～1%=0,1～10%=1,10～50%=2,50～80%=3,80～100%=4

B is the color intensity grade of the positive cells:0(negative),1 (weakly positive),2 (positive),3(strongly positive)

Statistical analysis

SPSS 20.0 software was used for statistical data analysis.The measurement data were expressed as χ ±s,and repeated measurements were analyzed by Repeated Measures.Differences among groups were compared through analysis of variance or non-parametric tests,and differences between groups were analyzed with theLSDmethod.WhenP< 0.05,the differences were considered statistically significant.

Results

Effects of the Qingre Yangyin Chushi pill on the joints of rats in each group

Before modeling,the rats of each group had basically the same ankle circumference; the difference was not statistically significant (P> 0.05),but,rather,comparable.At 6 h after modeling,joint swelling,decreased activity,foot licking,and other phenomena appeared in the model rats of all groups except the BC group.In addition,the swelling level of rats in each group increased compared to before modeling,though the difference was not statistically significant (P>0.05).

At 12 h after modeling,the ankle swelling index in each group had increased compared to the BC group,and the difference was statistically significant (P<0.05).Compared to the model group,the joint swelling index in the PC group was significantly lower (P<0.05) but showed no significant difference with the other drug groups (P> 0.05).Compared to the PC group,the joint swelling index was slightly higher in the MSD1,MSD2,and MSD3 groups,but the difference did not reach statistical significance (P>0.05).

At 24 h after modeling,the ankle swelling index in each group had increased compared to the BC group,and the difference was statistically significant (P<0.05).Compared with the model group,the joint swelling index of each drug group showed no significant difference (P> 0.05).Likewise,compared to the PC group,the swelling index showed no significant difference with the MSD1,MSD2,or MSD3 group (P> 0.05).To the BC group,the ankle swelling index showed no significant difference in the MSD2 group (P> 0.05) at 48 h after modeling,but it was higher in the other groups,and the difference was statistically significant (P< 0.05).Compared to the model group,the ankle swelling index was lower in each drug group,and the difference was statistically significant (P< 0.05).Compared with the PC group,the ankle swelling index was significantly lower in the MSD2 group,and the difference was statistically significant (P=0.01); the MSD1 and MSD3 groups each had a joint swelling index similar to the PC group(P> 0.05),as shown in Table 2.HE staining of ankle synovial tissue is shown in Figure 1.

Through the period of continuous measurement,compared with the BC group,clear ankle swelling was noted in rats of each group.Arthritis in rats in the BC group (without drug intervention) reached a peak within 48 hours; the ankle swelling index of rats in each drug group reached a peak within 24 hours.However,it lowered with prolonged treatment beyond 24 hours,especially in the MSD2 group,indicating that arthritis was improved in each drug group,but the efficacy was more obvious in the MSD2 group.

Table 2 Effects of the Qingre Yangyin Chushi pill on the ankle swelling index of rats (χ±s,n=8)

Effects of the Qingre Yangyin Chushi pill on inflammatory cytokines of rats

Gouty arthritis is a systemic inflammatory response.With little synovial fluid in the ankle of rats,collection of the joint washing fluid may cause significant dilution.IL-1β,IL-18,and TNF-α are downstream inflammatory cytokines,so their levels in blood can also reflect the severity of arthritis.

Effect on IL-1β level in each group.At 72 hours after modeling,the expression level of IL-1β was increased in the model and MSD1 groups,and was significantly decreased in the PC,MSD2,and MSD3 groups,compared with the BC group.The differences among the groups were statistically significant (P< 0.05).The expression was significantly decreased in each drug group compared with the model group,and the differences among the groups were statistically significant (P< 0.05).The expression was similar in the MSD3 group compared with the PC group,but the difference was not statistically significant (P=0.35).The expression was decreased significantly (P< 0.05)in the MSD2 and MSD3 groups and more significantly(P=0.01) in the MSD2 group.

Effect on IL-18 level in each group.Compared with the BC group,the expression level of IL-18 was not significantly different (P< 0.05) in the MSD3 group.It was increased (P< 0.05) in the model group and MSD1 groups and was decreased (P< 0.05) in the PC and MSD2 groups.The expression in each drug group was decreased compared with the model group,and the difference was statistically significant (P< 0.05).Compared with the PC group,the expression was similar (P=0.11) in the MSD3 group,increased (P=0.01) in the MSD1 group,and decreased (P=0.00) in the MSD2 group.

Effect on TNF-α level in each group.Due to the poor normality of the data,SPSS software was used to analyze the original data after lg conversion.Compared with the BC group,the expression level of TNF-α was increased in the model and MSD1 groups and decreased in the other drug groups.The differences among the groups were statistically significant (P< 0.05).As shown in Table 3,the expression was similar (P=0.20) in the MSD3 group,increased (P< 0.05) in the MSD1 group,and decreased (P=0.00) in the MSD2 group compared with the PC group.

The above results showed that the expression levels of IL-1β,IL-18,and TNF-α were significantly decreased in the MSD2 group compared with those in the BC and model groups,and that the inhibition was more significant in the MSD2 group than in the PC group.

Effects of the Qingre Yangyin Chushi pill on the mRNA expression of NLRP3/GSDMD pathway proteins

The mRNA expression of NLRP3 protein in each group.The NLRP3 mRNA level was significantly increased in the model and MSD1 groups compared with the BC group,and the difference was statistically significant (P< 0.05).No significant difference (P>0.05) was observed among the other drug groups,and no significant difference (P> 0.05) was observed between the model and MSD1 groups.The expression level was decreased in the other drug groups,and the differences among the groups were statistically significant (P< 0.05).Compared with the PC group,the NLRP3 mRNA level was significantly increased (P< 0.05) in the MSD1 group,showed no significant difference (P=0.06) in the MSD3 group,and was decreased in the MSD2 group.These differences were statistically significant (P=0.04).

The mRNA expression of Capase-1 protein in each group.The expression level of Capase-1 mRNA was significantly increased (P< 0.05) in the model,PC,and MSD1 groups,and showed no significant difference (P> 0.05) in the MSD2 and MSD3 groups compared with that in the BC group.Compared with the model group,no significant difference (P> 0.05)was observed in the PC and MSD1 groups.The ex pression levelofCapase-1mRNAwas increasedin the MSD2 and MSD3groups,and the difference wasstatistically significant (P< 0.05).The expression showed no significant difference (P> 0.05) in the MSD1 group compared with the PC group,was decreased in both the MSD2 (P=0.01) and MSD3 (P=0.02) groups.The decrease was more significant in the MSD2 group,and the differences were statistically significant.

Table 3 Effects of Qingre Yangyin Chushi pill on the content of inflammatory cytokines in synovial tissue of rat (χ±s,n=8) (pg·mL?1)

The mRNA expression of ASC protein in each group.The expression of ASC mRNA showed no significant difference (P> 0.05) in the MSD3 group,was increased in the model and MSD1 groups,and was decreased in the PC and MSD2 groups compared with the BC group.The differences among the groups were statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group,and the difference was statistically significant(P< 0.05).The expression showed no significant difference (P> 0.05) in the MSD3 group,was increased (P< 0.05) in the MSD1 group,and was decreased (P=0.01) in the MSD2 group compared with the PC group.

The mRNA expression of GSDMD protein in each group.The mRNA expression level of GSDMD was increased in the model and MSD1 groups and decreased in the other drug groups compared with the BC group.The differences were statistically significant(P< 0.05).The expression was decreased in each drug group compared with the model group.This difference was statistically significant (P< 0.05).As shown in Table 4,the expression was decreased in the MSD2 group compared with the PC group,and the difference was statistically significant (P=0.01).That the mRNA expression level of each key protein was decreased in the MSD2 group compared with the BC and model groups.Compared with the PC group,the Qingre Yangyin Chushi pill medium-dose group had more significant inhibition on the mRNA expression of each key protein.

Effects of the Qingre Yangyin Chushi pill on the key protein expression levels of the NLRP3/GSDMD pathway

Expression of NLRP3 protein in each group.The expression of NLRP3 protein showed no significant difference (P> 0.05) in the MSD2 group and was increased in the other groups compared with the BC group.The differences were statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group.This difference was statistically significant (P< 0.05).The expression was increased in the MSD1 and MSD3 groups compared with the PC group,and the difference was statistically significant (P< 0.05).The expression was similar in the MSD2 group,and this difference was not statistically significant (P=0.14).

Expression of Capase-1 protein in each group.The expression of Capase-1 protein was decreased in the MSD2 group compared with the BC group.The difference was not statistically significant (P> 0.05).The expression in the other groups was increased,and the difference was statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group,and the difference was statistically significant (P< 0.05).The expression was decreased in the MSD2 group compared with the PC group,and this difference was statistically significant (P=0.02).The expression was similar in the MSD1 and MSD3 groups,and the difference was not statistically significant (P> 0.05).

Expression of ASC protein in each group.The expression was similar in the MSD2 group compared with the BC group,and the difference was not statistically significant (P> 0.05).The expression was increased in the other groups,and the difference was statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group,and the difference was statistically significant(P< 0.05).The expression was decreased in the MSD2 group compared with the PC group,and the difference was statistically significant (P=0.01).The expression was increased in the MSD1 and MSD3 groups,and the difference was statistically significant (P< 0.05).

Expression of GSDMD protein in each group.Compared with the BC group,the MSD2 group had no significantly different (P> 0.05) expression of GSDMD protein.The expression in the other groups was increased,and the difference was statistically significant (P< 0.05).

Table 4 Effects of Qingre Yangyin Chushi pill on mRNA expressions of NLRP3,Caspase-1,ASC,GSDMD in synovial tissue of rat (χ±s,n=8)

Effects of the Qingre Yangyin Chushi pill on the key protein expression levels of the NLRP3/GSDMD pathway

Expression of NLRP3 protein in each group.The expression of NLRP3 protein showed no significant difference (P> 0.05) in the MSD2 group and was increased in the other groups compared with the BC group.The differences were statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group.This difference was statistically significant (P< 0.05).The expression was increased in the MSD1 and MSD3 groups compared with the PC group,and the difference was statistically significant (P< 0.05).The expression was similar in the MSD2 group,and this difference was not statistically significant (P=0.14).

Expression of Capase-1 protein in each group.The expression of Capase-1 protein was decreased in the MSD2 group compared with the BC group.The difference was not statistically significant (P> 0.05).The expression in the other groups was increased,and the difference was statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group,and the difference was statistically significant (P< 0.05).The expression was decreased in the MSD2 group compared with the PC group,and this difference was statistically significant (P=0.02).The expression was similar in the MSD1 and MSD3 groups,and the difference was not statistically significant (P> 0.05).

Expression of ASC protein in each group.The expression was similar in the MSD2 group compared with the BC group,and the difference was not statistically significant (P> 0.05).The expression was increased in the other groups,and the difference was statistically significant (P< 0.05).The expression was decreased in each drug group compared with the model group,and the difference was statistically significant(P< 0.05).The expression was decreased in the MSD2 group compared with the PC group,and the difference was statistically significant (P=0.01).The expression was increased in the MSD1 and MSD3 groups,and the difference was statistically significant (P< 0.05).

Expression of GSDMD protein in each group.Compared with the BC group,the MSD2 group had no significantly different (P> 0.05) expression of GSDMD protein.The expression in the other groups was increased,and the difference was statistically significant (P< 0.05).The expression in each drug group was decreased compared with the model group,and the difference was statistically significant (P<0.05).The expression in the MSD2 group was decreased compared with the PC group,and the difference was statistically significant (P=0.01).As shown in Table 5 and Figure 2,the expressions in the MSD1 and MSD3 groups were similar,and had no statistical significance (P< 0.05).

The above results indicate that the expression of pathway key proteins in the MSD2 group was significantly lower than in the BC and model groups,and that it was inhibited more significantly compared with the PC group.

Effects of the Qingre Yangyin Chushi pill on IHS scores of key proteins in the NLRP3/GSDMD pathway.

Expression of NLRP3 in each group.The expression level of NLRP3 was higher in the model and MSD1 groups and lower in the other drug groups compared with the BC group.The difference was statistically significant (P< 0.05).The expression was lower in each drug group than in the model group,and the difference was statistically significant (P< 0.05).The expression was significantly lower in the MSD2 group compared with the PC group,and the difference was statistically significant (P=0.01).The expression was higher in the MSD1 group,and the difference was statistically significant (P< 0.05).The expression was similar in the MSD3 group,and the difference was not statistically significant (P> 0.05).

Expression of Capase-1 in each group.Compared with the BC group,the expression was higher in the model group and lower in the MSD2 group,and the difference was statistically significant (P< 0.05).The expression was similar in the other groups,and the difference was not statistically significant (P> 0.05).The expression was lower in each drug group than in the model group,and the difference was statistically significant (P< 0.05).The expression was significantly lower in the MSD2 group compared with the PC group,and the difference was statistically significant (P=0.01).The expression was higher in the MSD1 group,and the difference was statistically significant (P<0.05).The expression was similar in the MSD3 group,and the difference was not statistically significant (P>0.05).

Expression of ASC in each group.The expression level of ASC was significantly higher in the model group and significantly lower in the MSD2 group compared with the BC group.This difference was statistically significant (P< 0.05).The expression was similar in the other groups,and the difference was not statistically significant (P> 0.05).Compared with the mode group,the expression was similar (P> 0.05) in the MSD1 group and lower in the other drug groups.The difference was statistically significant (P< 0.05).The expression was significantly lower in the MSD2 group compared with the PC group,and the difference was statistically significant (P=0.01).The expression was higher in the MSD1 group,and the difference was statistically significant (P< 0.05).The expression was similar (P> 0.05) in the MSD3 group.

Expression of GSDMD in each group.Compared with the BC group,the expression of GSDMD was higher in the model and MSD1 groups and lower in the MSD2 group.The difference was statistically significant (P< 0.05).The expression was similar (P>0.05) in the other groups.Compared with the model group,the expression was similar in the MSD1 group,and the difference was not statistically significant (P>0.05).The expression was lower in the other drug groups,and the difference was statistically significant(P< 0.05).Compared with that in the PC group,the expression was significantly lower in the MSD2 group,and the difference was statistically significant (P=0.01).The expression was higher in the MSD1 group,and the difference was statistically significant (P<0.05).The expression was similar (P> 0.05) in the MSD3 group,as shown in Table 6.

The above results show that the MSD2 group had significantly lower expression of key proteins than the BC and model groups and stronger inhibition than the PC group.

Table 5 Effects of Qingre Yangyin Chushi pill on expressions of NLRP3,Caspase-1,ASC and GSDMD in synovial tissue of rat (χ±s,n=8)

Table 6 Effects of Qingre Yangyin Chushi pill on IHS scores of NLRP3,Caspase-1,ASC,GSDMD in synovial tissue of rat (χ±s,n=8)

Discussion

Gout is a crystal-related arthropathy caused by the deposition of sodium urate monohydrate.It is directly associated with the hyperuricemia caused by purine metabolic disorder and/or decreased uric acid excretion.The main clinical manifestations of gout are recurrent acute arthritis,irreversible peripheral joint injury,subcutaneous tophus,gouty nephropathy,and lithangiuria,which may eventually result in abnormal functions of important organs [11].According to data from the Chinese Rheumatism Data Center,79% of patients can't work normally because of pain and hospitalization,and their work and quality of life are seriously affected during gout attacks.Therefore,the economic and social losses caused by gout cannot be ignored [12].

With phagocytosis of monosodium urate crystals,neutrophil plays a central role in the spread of acute inflammation during gout attacks,but the recruitment of neutrophils to joints requires early local cell and liquid phase events in blood vessels of the synovium and joints.These events initiate the inflammations mediated by the innate immune system,including activation of the phagocytes settled in the synovium and the NOD-like receptor protein 3 (NLRP3)inflammasome,which leads to the processing of IL-1 precursor cells and the activation of key cytokine IL-1β,thus causing a severe inflammatory response.Pyroptosis is an inflammatory programmed cell death discovered by Cookson et al.[13,14] in 2001.With Caspase-1 as the core link and GSDMD protein as the cutting substrate,it is a classical pathway of pyroptosis.Activated by the NLRP3 inflammasome after sensing pathogen signals,Caspase-1 is one of the most important pathways for innate immunity of cytoplasm[15].

After the urate crystals are precipitated or the tophus is disintegrated in patients with gouty arthritis,the activated NLRP3 inflammasome and Caspase-1 can mediate the pyroptosis of macrophages in synovial tissue,which is manifested as the rupture of cell membrane and the release of inflammatory cytokines,such as IL-1β,synergistically amplify the strong inflammatory response,and result in the typical symptoms of gouty arthritis,such as fever,joint swelling,and pain [16,17].The NLRP3/GSDMD pyrolysis pathway is strongly associated with the acute inflammation of gout.The experimental results showed that the model group had significantly higher expression levels of main proteins and mRNA in the NLRP3/Caspase-1 pathway and higher contents of inflammatory cytokines,such as IL-1β,TNF-α,and IL-18,than the BC group.These results are consistent with the pathogenesis of gouty arthritis,which indicates the successful modeling,as well as the important role of the NLRP3/GSDMD pathway in acute inflammations of gout.As a classical drug for controlling gouty arthritis,colchicine can inhibit the expression of NLRP3.According to results of this study,the colchicine group has significantly lower expression levels of related proteins in the NLRP3/GSDMD pathway than the model group.The arthritis score in the colchicine group was significantly lower than in the model group,indicating that colchicine can control the symptoms of gouty arthritis.

Pharmacological studies of the Qingre Yangyin Chushi pill show that this drug can reduce the foot swelling and arthritis index of type II collagen-induced arthritis in rats and lower the IL-1,IL-6,and TNF-α levels in serum,suggesting that the mechanism of this drug may be associated with lowering the abnormally elevated cytokines,reducing the production of proinflammatory cytokines,and regulating the broken balance between proinflammatory and anti-inflammatory cytokines [18].In addition,the Qingre Yangyin Chushi pill can increase the pain threshold of mice and inhibit the inflammatory ear swelling and foot swelling in mice,which may be related to the reduction of inflammatory mediators.The research team has carried out clinical studies on the treatment of gout with this drug [19,20],and the results show no significant difference with the colchicine group in controlling the joint pain,liver and kidney functions,white blood cells,erythrocyte sedimentation rate,and C-reactive protein.The Qingre Yangyin Chushi pill has been demonstrated to have good effects on controlling inflammation of joints.According to this study,the arthritis and joint swelling scores in each dose group of the Qingre Yangyin Chushi pill were lower than those in the model group,but similar to those in the colchicine group.The content of inflammatory cytokines,such as IL-1β,IL-18,and TNF-α in each dose group was lower than in the model group.It was similar between the low-dose group and the colchicine group,and it was significantly lower in the medium-and high-dose groups than in the colchicine group.In terms of regulating the NLRP3/GSDMD pathway,the medium-dose group showed significant inhibition on the expression of related proteins in this pathway compared with the colchicine group,whereas the lowand high-dose groups were similar to the colchicine group.The results showed that the effect of the Qingre Yangyin Chushi pill on the inflammatory pathway was not dose-dependent,which may indicate that this drug has been saturated with the drug receptor at a certain dose.Therefore,even if the drug dose is increased,the anti-inflammatory effect will not be increased,which is similar to the phenomenon observed in clinical applications.

Conclusion

In summary,this study demonstrates that the Qingre Yangyin Chushi pill can inhibit the activation of NLPR3 inflammasomes,reduce the production of GSDMD protein,regulate the pyrolysis,and reduce the expression of inflammatory cytokines,thus mitigating the inflammation of joints.The anti-inflammatory effect of the Qingre Yangyin Chushi pill may be achieved through the NLRP3/GSDMD pathway.This study provides an important foundation for further studies on the action mechanism of this drug,as well as a biological basis for the treatment of gouty arthritis with this drug.