| With the change of life style,the incidence of chronic prostatitis(CP)is increasing,among which type Ⅲ prostatitis is the most common.Type Ⅲ prostatitis patients with pelvic pain and discomfort,abnormal urination as the main symptoms,and even lead to mental and psychological abnormalities,sexual dysfunction and other problems,seriously affecting the quality of life of men.Western medicine is often treated with α-receptor blockers,antibiotics and non-steroidal anti-inflammasome drugs,but the improvement of clinical symptoms is not ideal,or although it is relieved,it is easy to occur repeatedly after drug withdrawal,resulting in low satisfaction with the treatment of type Ⅲ prostatitis.The Department of Andrology,Xiyuan Hospital of China Academy of Chinese Medical Sciences,used Guihuang Formula to treat type Ⅲ prostatitis with dampness-heat and blood stasis syndrome.The previous study showed that Guihuang Formula could significantly reduce the National Institute of Health-chronic prostatitis symptom index(NIH-CPSI)score and TCM symptom score of patients treated with Guihuang Formula for 6 weeks.However,the sample size of the previous study is small,so this study we ues RCT clinical study to further clarify the efficacy and safety of Guihuang Formula in the treatment of type Ⅲ prostatitis with dampness-heat stasis syndrome.Previous animal experiments showed that Guihuang Formula significantly reduced the pathological inflammation of prostate tissue and the expression of pro-inflammasome cytokines in rats with type Ⅲ prostatitis,which was related to the inhibition of PI3K and NF-κB.NLRP3 inflammasome,as an important protein regulating inflammasome response,have been reported to increase the expression of NLRP3 inflammasome in prostate of CP rats.Activation of NLRP3 inflammasome requires two steps:one is the assembly process induced by NLRP3 inflammasome,which is related to NF-κB,and the other is the assembly activation process of NLRP3 inflammasome,which is related to ROS release and K+efflux.Previous studies have shown that Guihuang Formula can inhibit the expression of NF-κB,so does Guihuang Formula regulate the priming of NLRP3 inflammasome by inhibiting NF-κB?At the same time,can Guihuang Formula inhibit the activation of NLRP3 inflammasome?Therefore,in this study,animal experiments and cell experiments were conducted to verify whether Guihuang Formula can play a role in the treatment of type Ⅲ prostatitis by inhibiting the priming and activation process of NLRP3 inflammasome.However,activation of NLRP3 inflammasome can not only cause the release of inflammasome factors,but also cause pyroptosis.Although cell death plays an important role in many chronic inflammasome diseases,but the role in type Ⅲprostatitis has not been fully clarified.Therefore,this study puts forward the hypothesis that Guihuang Formula may inhibit the activation and priming induced by NLRP3 inflammasome,then inhibit the activation of Caspase-1,prevent the cleavage of GSDMD,reduce the release of pro-inflammasome cytokines IL-1β and IL-18,and play a role in the treatment of type Ⅲ prostatitis.Objective1.Through RCT clinical study,to further clarify the efficacy and safety of Guihuang Formula in the treatment of dampness-heat and blood stasis syndrome of type III prostatitis,so as to provide evidence-based basis for the popularization and application of Guihuang Formula.2.Through animal and cell experiments,focusing on NLRP3 inflammasome,we studied the possible mechanism of Guihuang Formula in the treatment of type Ⅲprostatitis by regulating the activation of NLRP3 inflammasome and inhibiting pyroptosis.Method1.Clinical study:in this randomized,controlled trial,120 patients with type Ⅲprostatitis with dampness-heat stasis syndrome were enrolled.All of them were admitted to the male outpatient clinic of The Department of Andrology,Xiyuan Hospital of China Academy of Chinese Medical Sciences,from June 2021 to january 2022.All of them met the discharge standard of damp-heat stasis syndrome of type Ⅲprostatitis,and passed the ethical review,ethical batch number:2021XLA0482.Random numbers were generated by SAS and randomly divided into two groups at 1:1.60 patients in the experimental group were treated with Guihuang Formula(drug composition:Angelica sinensis,Cortex Phellodendri,turmeric,Angelica dahurica,frankincense,myrrh,honeysuckle,Hedyotis diffusa,plantain,tangerine peel).Directions:1 bag twice a day.Sixty patients in the control group were given tamsulosin hydrochloride sustained release capsule(Chinese medicine H20050392).Directions:take one tablet once a day before going to bed.The patients were treated continuously for 6 weeks and followed up for 4 weeks.The main curative effect index uses the recognized NIH-CPSI score,the secondary curative effect index is the TCM symptom score and expressed prostatic secretion.The safety evaluation indexes were blood,urine routine,liver function(ALT,AST),renal function(BUN,Cr)and electrocardiogram.2.In the animal experiment,50 SD rats were randomly divided into blank group(n=10),model group(n=10),Guihuang Formula low dose group(n=10),middle dose group(n=10)and high dose group(n=10).Except the blank group,the other 4 groups were used to establish the rat model of autoimmune type III prostatitis.After successful establishment of the model,the blank group and the model group were perfused with normal saline,and the low,middle and high dose groups were given 4.9g/kg·d,9.8g/kg·d and 19.6g/kg·d respectively.Euthanasia was performed after 30 days,and the samples were detected.The infiltration of inflammasome cells in prostate tissue was observed by HE staining,the levels of pro-inflammasome cytokines IL-1β and IL-18 in serum were detected by ELISA method,and the levels of MDA,T-SOD and GSH-Px in serum of rats were detected by biochemical method,the expression of ROS in prostate tissue was detected by fluorescence,the expression of NLRP3 in prostate tissue was detected by immunohistochemistry,the expression of GSDMD in prostate tissue was detected by immunofluorescence,and the expression of NLRP3,Caspase-1 and GSDMD protein was detected by Western Blot.3.In cell experiment,human normal prostate epithelial cells(RWPE-1 cells)were divided into blank group,model group,Guihuang Formula group and NLRP3 inhibitor group.Except the blank group,the other three groups were stimulated by LPS100ng/mL for 4 hours,and 30min was stimulated by ATP 5mM to establish the model of pyroptosis.After successful modeling,blank group and model group were given blank serum,Gui huang Formula group was added with 6.25ug/ml containing drug serum of Guihuang Formula,NLRP3 inhibitor group was added MCC950 on the basis of model group.The drug-containing serum components of Guihuang Formula were determined by UPLC-Q/TOF-MS,the morphology of cell scorch was observed by transmission electron microscope,PI uptake was detected by flow cytometry,LDH level in cell supernatant was detected by biochemistry,Caspase-1 expression was detected by flow cytometry,Tunel staining was detected by flow cytometry,ROS was detected by flow cytometry,expression of MDA,T-SOD and GSH-Px was detected by biochemistry,and IL-1β and IL-18 levels in cell supernatant were detected by ELISA method.The protein expressions of NLRP3,Caspase-1 and GSDMD were detected by Western Blot.Results1.Clinical study(1)A total of 120 subjects were enrolled in this clinical study,including 60 cases in the experimental group and 60 cases in the control group.At the end of the study,there were 8 shedding cases,including 3 cases in the experimental group and 5 cases in the control group.2 cases were excluded,1 case in the experimental group and 1 case in the control group.Finally,a total of 110 cases were in accordance with the protocol set(PPS),including 56 cases in the experimental group and 54 cases in the control group.(2)Compared with the control group before and after treatment,the NIH-CPSI score of the test group decreased from 28.34±9.23 to 6.78±3.53 after 6 weeks of treatment,and the NIH-CPSI score of the control group decreased from 27.81±8.28 to 14.48±4.27 after treatment.The TCM symptom score decreased from(19.21±2.58)to(5.11±1.09)in the test group and from(17.49±2.21)to(11.28±1.94)in the control group.The comparison between the two groups showed that the total score of NIH-CPSI,pain score,voiding score,quality of life influence score and TCM symptom score were decreased in both groups.(P<0.05).The stratified statistical results of ⅢA and ⅢB are consistent with the overall results.(3)Compared with the control group at the same time,except that there was no significant difference in the voiding score between the experimental group and the control group at the second week of treatment,the total NIH-CPSI score,pain score,micturition score,quality of life score,TCM symptom score and the total effective rate of disease in the experimental group were better than those in the control group at the 4th,6th and 10th week.(P<0.05).The stratified statistical results of ⅢA and ⅢB are consistent with the overall results.(4)The expressed prostatic secretions(EPS)results showed that WBC decreased and lecithin body increased in the two groups after treatment.Compared with the control group,the experimental group decreased the number of WBC and increased the number of lecithin body.(5)Safety evaluation:there was no obvious abnormality in hematuria routine,liver and kidney function and ECG in the two groups after treatment.In the experimental group,one case had gastric distension after taking medicine on an empty stomach and disappeared after taking medicine after meal,and the other case had diarrhea because he was not given warm medication.In the control group,3 patients developed transient hypotension after taking the medicine,and the discomfort disappeared after taking it before going to bed.No obvious adverse reactions were found in other patients during the treatment.2.Animal experiment(1)The results of HE staining showed that the infiltration of inflammasome cells in the prostate tissue of the model group was more obvious than that of the blank group(P<0.001).Compared with the model group,the low,middle and high groups of Guihuang Formula could reduce the infiltration of inflammasome cells in prostate tissue and significantly improve the histopathological changes of prostate in rats with type Ⅲ prostatitis(P<0.001).(2)The results of ELISA showed that the levels of serum pro-inflammasome cytokines IL-1β and IL-18 in the model group were significantly higher than those in the blank group(P<0.001),and the levels of IL-1β and IL-18 were significantly decreased after intervention by Guihuang Formula(P<0.001).(3)The results of biochemical test showed that the level of serum MDA in the model group increased,and the level of MDA decreased significantly after the intervention of Guihuang Formula(P<0.001),while the levels of T-SOD and GSH-Px in the model group decreased,and the level of T-SOD and GSH-Px increased significantly after the intervention of Guihuang Formula(P<0.001).(4)ROS fluorescence positive staining of prostate in the model group,after the intervention of Guihuang Formula,the positive staining of ROS decreased,especially in the high dose group.(5)The immunohistochemical results of NLRP3 showed that the expression of NLRP3 in the prostate tissue of the model group increased,and the expression of NLRP3 decreased significantly after the intervention of Guihuang Formula.(6)The results of GSDMD immunofluorescence showed that the expression of GSDMD in the prostate tissue of the model group increased,and the fluorescence intensity of GSDMD decreased significantly after the intervention of Guihuang Formula.(7)The results of Western Blot showed that the protein expression of NLRP3,Caspase-1 and GSDMD in the prostate tissue of the model group was significantly higher than that of the blank group(P<0.001),and the expression of NLRP3,Caspase-1 and GSDMD protein was significantly decreased after the intervention of Guihuang Formula,especially in the high dose group(P<0.001).3.Cell experiment(1)The serum components of Guihuang Formula were determined by UPLC-Q/TOF-MS,including Ursolic acid,Natsudaidain,Olibanumols 1,β-Boswellic acid,Nodakenetin and so on.(2)CCK-8 detection showed that the best concentration of Guihuang Formula containing serum on RWPE-1 cells was 6.25ug/ml.(3)Under light microscope,there were more dead cells,irregular cell shape,more cytoplasmic impurities and unclear nucleus in the model group,while the floating dead cells in the Guihuang Formula group and the inhibitor group were less than those in the model group,and the cytoplasm was clear and the nucleus was clear.(4)The transmission electron microscope showed that in the model group,the cells became large and round,the cell membrane ruptured,bubble-like protrusions and membrane pores were found,which showed the typical morphology of cell death.(5)PI uptake was detected by flow cytometry.The PI uptake rate of RWPE-1 cells in the model group was higher than that in the blank group,and the PI uptake rate in the model group was significantly higher than that in the blank group(P<0.001).The PI uptake rate in Guihuang Formula group and inhibitor group was significantly lower than that in model group(P<0.001).(6)The release of LDH in the model group increased,indicating that the release of cell rupture contents increased(P<0.001).After the intervention of Guihuang Formula drug-containing serum and MCC950,the release of LDH was inhibited,which was significantly different from that in the model group(P<0.001).(7)The expression of Caspase-1 in the model group was significantly higher than that in the blank group(P<0.001).The expression of Caspase-1 in the Guihuang Formula group and inhibitor group was significantly lower than that in the model group(P<0.001).(8)TUNNEL staining,the positive cells of TUNNEL staining in the model group were more than those in the blank group,while those in the Guihuang Formula group and inhibitor group were significantly decreased.(9)ROS detected by flow cytometry showed that the level of ROS in the model group was significantly higher than that in the blank group(P<0.001),while the level of ROS in the Guihuang Formula and inhibitor group was significantly lower than that in the model group(P<0.001).(10)Biochemical detection of peroxidation index MDA,the level of MDA in the model group was significantly higher than that in the blank group(P<0.001),and the level of MDA in the Guihuang Formula group and inhibitor group was significantly lower than that in the model group(P<0.001).T-SOD and GSH-Px detection showed that compared with the blank group,the levels of T-SOD and GSH-Px in the model group were significantly lower,T-SOD and GSH-Px were significantly lower(P<0.001),T-SOD in the Guihuang Formula group and inhibitor group was significantly higher than that in the model group(P<0.001),and GSH-Px in the Guihuang Formula group and inhibitor group was significantly higher than that in the model group(P<0.01).(11)The results of ELISA detection showed that the levels of IL-1β and IL-18 in the supernatant of the model group were significantly higher than those in the blank group(P<0.001),while the levels of IL-1β and IL-18 in the Guihuang Formula group and inhibitor group were significantly lower than those in the model group(P<0.001).(12)Western Blot detection:compared with the blank group,the protein expression levels of NLRP3,Caspase-1 and GSDMD of NLRP3 inflammasome in the model group increased(P<0.05);while the protein expression levels of NLRP3,Caspase-1 and GSDMD decreased in the Guihuang Formula group and the inhibitor group.(P<0.05).Conclusion1.Clinical research shows that Guihuang Formula can significantly reduce the total NIH-CPSI score,pain score,voiding score,quality of life score and TCM symptom score of patients with damp-heat stasis syndrome of type Ⅲ prostatitis,decrease the number of WBC and increase the number of lecithin bodies,and the curative effect is better than that of tamsulosin hydrochloride sustained release capsule.Guihuang Formula has no serious adverse reactions before and after treatment,and has high safety,so it is worth popularizing and applying in clinic.2.Animal experiments showed that Guihuang Formula could significantly reduce the pathological inflammation of prostate tissue and oxidative stress injury in rats with type III prostatitis.At the same time,Guihuang Formula can inhibit the activation of assembly and assembly induced by NLRP3 inflammasome,then inhibit the activation of Caspase-1,prevent the cleavage and cleavage of GSDMD,inhibit the occurrence of cell scorch,reduce the inflammasome cascade reaction caused by the release of pro-inflammasome cytokines IL-1β and IL-18,and play a role in the treatment of typeⅢ prostatitis.3.Cell experiments showed that RWPE-1 pyroptosis model could be prepared by the combination of LPS and ATP stimulation,and Guihuang Formula drug-containing serum could inhibit the scorch death of RWPE-1 cells by inhibiting the activation of NLRP3 inflammasome.In conclusion,this study clarify the efficacy and safety of Guihuang Formula in the treatment of dampness-heat and blood stasis syndrome of type Ⅲ prostatitis,the mechanism of Guihuang Formula in the treatment of type Ⅲ prostatitis by regulating the activation of NLRP3 inflammasome and inhibiting pyroptosis. |
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