Influence Of Qiantongding Decoction On Chronic Prostatitis-induced Pain And P38MAPK Signal Transduction Pathways Of Spinal Microglia In Models

Objective:To observe the effectiveness and safety of Qiantongding Decoction on the Type Ⅲ chronic prostatitis(CP)-induced pain classified with phenotypic classification system(UPOINT,urinary symptoms,psychosocial,organ-specific,infection,tenderness of pelvic floor skeletal muscles).With the Type Ⅲ CP animal models established,the influence of Qiantongding Decoction on p38MAPK signal transduction pathways of spinal microglia was observed,which further explores the possible mechanisms and action targets of Qiantongding Decoction on the Type Ⅲ CP-induced pain.Methods:Clinical study:In total 120 cases of Type Ⅲ CP-induced pain classified with UPOINT from the Andrology Department,Shenzhen Traditional Chinese Medicine Hospital,from January 2015 to January 2016 were randomized into treatment group(G1),control Ⅰ group(G2)and control Ⅱ group(G3),with 40 cases in each.In Gl,Qiantongding Decoction was orally applied;in G2,celecoxib capsule was orally used;and in G3,celecoxib capsule plus Qiantongding Decoction was orally taken,which lasted for continuous 1 month.Before and after treatments,the therapeutic effects of all the cases enrolled were assessed with National Institutes of Health-Chronic Prostatitis Symptom Index(CPSI)and CP syndrome differentiation scoring.The indexes such as tumor necrosis factor a(TNF-a),interleukin 10(IL-10),cyclooxygenase 2(cox 2)in the prostatic fluid of patients were detected with the Enzyme Linked Immunosorbent Assay(ELISA),and all the adverse reactions during the treatments were recorded.Animal experiments:A total of 80 six-week-old,male,specific pathogen-free Sprague-Dawley(SD)rats,weighing 150 g were housed and allowed free access to food and water for seven days,then they were randomized into normal group(Group A),sham modeling group(Group B),Qiantongding Group(Group C)and Water Group(Group D),with twenty rats in each.Rats in Group A were normally access to food.Rats in Group B were applied sham modeling,with normal saline injection(0.1ml)to the ventral lobes of the prostates.Rats in Group C and Group D were applied Freund’s complete adjuvant(FCA)injection(0.1ml)to the ventral lobes of the prostates to establish CP models.From the fifth day following the operations on,rats in Group A,Group B and Group D were intragastrically administered 1 ml/100g water,and rats in Group C were intragastrically administered 1 ml/100g Qiantongding Solution,once daily.All the rats were weighed once a week,and the amount of drugs was accordingly changed.On the fifth,eleventh,eighteenth and thirty-fourth day following the surgeries,five rats in each group were radiated to the pelma with the 5mm-diameter light spot of heat radiation stimulus(SOW,12V)to determine Thermal/Yithdrawal Latency(TWL).After scarified,the livers,kidneys,prostates and L5-S2 medulla spinalis were removed.After washed with cold physiological saline,the livers,kidneys and the left ventral lobes of the prostates were fixed with 10%neutral formalin for twenty-four hours.Following desiccation,paraffin embedding and tissue slicing,the hematoxylin and eosin(HE)counterstaining was applied and observed under light microscopy.The right ventral lobe,s of the prostates and the L5-S2 medulla spinalis were cut and placed in the 1.5-ml sterile centrifuge tube and stored at-80℃atmosphere.The IBA-1 nRNA and p38MAPK mRNA protein expressions in the L5-S2 medulla spinalis of C.P rats were detected with real-time fluorescence quota PCR;the p38MAPK and pp38MAPK protein expressions in the L5-S2 medulla spinalis were detected with Western Blotting,and the levels of P substance,TNF-αand COX-2 in the medulla spinalis and the prostates were detected with ELESA methods.Results:Clinical study:A total of one hundred and twenty patients were enrolled in the study.Among them,five cases in Gl,six in G2 and five in G3 were dropped due to other treatments during the study,and the other one hundred and four cases were successfully finished.Before the study,there was no statistical difference of the age distribution,disease courses,CPSI scores,pain symptom scores,CP syndrome differentiation scores,and the levels of the TNF-α,IL-10 and COX-2 among the three groups(P>0.05),which indicated good baselines among the three groups and had comparability.After the study,compared with before treatments,the CPSI scores,pain symptom scores,CP syndrome differentiation scores,and the levels of the TNF-α and COX-2 in all the three groups were decrea,sed,and the level of IL-10 was increased,with significant difference(P<0.05).For the comparisons among the groups:①Compared with the G2,the CPSI scores,pain symptom scores,CP syndrome differentiation scores,and the levels of the TNF-α and COX-2 in G1 and G3 were decreased with significant difference(P<0.05),and the level of IL-10 was increased with significant difference(P<0.05).②There was no significant difference of all the indexes between G1 and G3(P>0.05).③The total effective rate in G1 was 87.50%(5 cases cured,21remarkably effective,11 effective and 3 ineffective);the total effective rate in G2 was 62.5%(3 cases cured,7remarkably effective,15 effective and 15 ineffective);the total effective rate in G3 was 95.00%(6 cases cured,22 remarkably effective,10 effective and 2 ineffective).There was significant difference of the clinical therapeutic effects among the three groups(P<0.05),with the G1 and G3 superior to G2.④ During the treatments,in Gl,2 cases suffered from abdominal distention,2 suffered from diarrhea,1 suffered from slight itching,with the adverse reaction rate as 10%;in G2,3 cases suffered from gastric uncomfortable,1 suffered from vertigo,1 suffered from slight itching,with the adverse reaction rate as 12.50%;in G3,1 cases suffered from abdominal distention,4 suffered from gastric uncomfortable,1 suffered from vertigo,with the adverse reaction rate as 15%.No serious adverse reaction occurred.All the patients suffering from adverse reactions continued the planned pharmaceutical application without any special management,and finally all the reactions disappeared.There was no significant difference of the adverse reaction rate among the three groups(P=0.4627>0.05).Animal experiments:All the SD rats were enrolled in the study.①During the anatomy with the naked eyes,the forms of the livers and kidneys in each group were generally normal,with the complete intense red capsules and without obvious necrosis,blutene chloaide,swelling,or remarkable changes of the texture.Under the light microscope,the forms of the livers and kidneys in each group were generally normal.②TWL value:There was no significant difference of the TWL values at all the time points between Group A and Group B(P>0.05).On the 5th day following surgeries,the TWL values in Group C and Group D were obviously decreased compared with those in Group A and Group B,with significant difference(P<0.05).In Group C,the TWL values on the 11th and 18th days following surgeries were gradually increased compared with that on the 5th day(P<0.05).On the 34th day following surgeries,there was no significant difference of the TWL values between Group A and Group B(P>0.05).In Group D,the TWL values on the 11th,18th,34th days following surgeries were slightly increased,without significant difference compared with that on the 5th day(P>0.05).③Observation with naked eyes:All the ventral lobes of the prostates in Group A and Group B were intense red,without any obvious edema,congestion or necrosis.In Group C,on the 5th day after surgeries,all the ventral lobes of the prostates showed edema,obvious congestion without local necrosis;on the 11th and 18th days,the edema and congestion alleviated compared with those on the 5th day without local necrosis;on the 34th day,there was no obvious edema,congestion or necrosis.In Group D,on the 5th and 11th days after surgeries,all the ventral lobes of the prostates showed edema,obvious congestion without local necrosis;on the 18th day,the edema and congestion alleviated without local necrosis;on the 34th day,there was no obvious edema,congestion or necrosis.④bservation under light microscope:The structures of all the prostates in Group A and Group B were normal,with the normal array of glands,pink secretion in the glandular cavity,and without edema in the mesenchyma or obvious lymphocytic infiltration.In Group C,on the 5th day following surgeries,the structures of the prostate glands were destroyed,with the glandular cavities expanded,and histocytes,inflammatory cells and degenerated and necrotic tissues filling in most of the glandular cavities.Most of the inflammatory cells were neutrophile granulocytes,with the mesenchyma edema,small vessels expanded and congested,and lymphocyte infiltration in the mesenchyma and glands,or with patchy distribution of the lymphocyte infiltration;on the 11th and 18th days,compared with those on the 5th day,the inflammation of the prostates obviously alleviated,accidentally lymphocyte infiltration occurred in the mesenchyma,or with patchy distribution of the lymphocyte infiltration;on the 34th day,the inflammation of the prostates obviously vanished,with the glandular epithelial cells slightly proliferated,rather regular array,small amounts of pink secretion in the glandular cavities,and small amounts of inflammatory cells diffusedly distributed in the mesenchyma.In Group D,on the 5th and 11th days following surgeries,the structures of the prostate glands were destroyed,with the glandular cavities expanded,and histocytes,inflammatory cells and degenerated and necrotic tissues filling in most of the glandular cavities.Most of the inflammatory cells were neutrophile granulocytes,with the mesenchyma edema,small vessels expanded and congested,and lymphocyte infiltration in the mesenchyma and glands,or with patchy distribution of the lymphocyte infiltration;on the 18th day,compared with those on the 5th day,the inflammation of the prostates obviously alleviated,accidentally lymphocyte infiltration occurred in the mesenchyma,or with patchy distribution of the lymphocyte infiltration;on the:34th day,the inflammation of the prostates obviously vanished,with the glandular epithelial cells slightly proliferated,rather regular array,small amounts of pink secretion in the glandular cavities,and small amounts of inflammatory cells diffusedly distributed in the mesenchyma.⑤ There was no significant difference of the IBA-1mRNA,p38MAPK mRNA and pp38MAPK protein expressions in the L5-S2 medulla spinalis and the levels of P substance,TNF-α and COX-2 in the prostatic fluid at different time points between Group A and Group B（P>0.05).⑥ There was no significant difference of the p38MAPK protein expression in the L5-S2 medulla spinalis at different time points among all the groups(P>0.05).⑦On the 5th day following surgeries,the expressions of IBA-1mRNA,p38MAPK mRNA and pp38MAPK proteins in the L5-S2 medulla spinalis and the levels of P substance,TNF-α and COX-2 in the L5-S2 medulla spinalis and prostatic fluid in Group C and Group D were increased,with significant difference compared with those in Group A and Group B(P<0.05).⑧ In Group C,on the 11th and 18th days following surgeries,the expressions of IBA-1mRNA,p38MAPK mRNA and pp38MAPK proteins in the L5-S2 medulla spinalis and the levels of P substance,TNF-α and COX-2 in the L5-S2 medulla spinalis and prostatic fluid were decreased,with significant difference compared with those on the 5th day after surgeries(P<0.05);on the 34th day after surgeries,there w-as no significant difference of the expressions of the IBA-lmRNA,p38MAPK mRNA and pp38MAPK proteins in the L5-S2 medulla spinalis and the levels of P substance,TNF-α and COX-2 in the L5-S2 medulla spinalis and prostatic fluid compared with those in Group A and Group B(P>0.05).⑨ In Group D,there was no significant difference of the expressions of the IBA-1mRNA,p38MAPK mRV A and pp38MAPK proteins in the L5-S2 medulla spinalis and the levels of P substance,TNF-a and COX-2 in the L5-S2 medulla spinalis on the 11th,18th and 34th days following surgeries compared with those on the 5th day after surgeries(P>0.05),and there was no significant difference of the levels of P substance and TNF-α in the prostates on the 34th day following surgeries in Group D compared with those in Group A and Group B(P>0.05).Conclusions:The present clinical study showed that Qiantongding Decoction could obviously decrease the CPSI scores,pain symptom scores and the CP syndrome differentiation scores of the Type Ⅲ CP-induced pain classified with UPOINT,improve the clinical symptoms and enhance the quality of life.The possible mechanisms may be correlated to the regulation of the levels of diverse inflammatory cytokines during the pain reactions,with safety.The present animal experiments showed as follows:① The CP modeling establishment with CFA injection to the ventral lobes of the prostates was successful.② For the prostatitis,the CP model rats has the tendency of self-healing.③ The continuity and generalization of CP-induceed pain is closely correlated to the secondary affection of the L5-S2 medulla spinalis,activation of the p38MAPK signal transduction pathways and the abnormal activation of microglia.④Qiantongding Decoction has the obvious effects of anti-inflammation and pain alleviation to the C.P model rats.⑤Qiantongding Decoction could inhibit the secretion of diverse endogenous inflammatory cytokines such as P substance,TNF-α and COX-2 in the prostates,reduce the signal transduction of p38MAPK signal transduction pathways in the L5-S2 medulla spinalis,inhibit the activation of the microglia,and further more reduce the secretions of the inflammatory cvtokines such as P substance,TNF-a and COX-2 in the L5-S2 medulla spinalis hence performing the actions of anti-inflammation and pain alleviation.⑥ Qiantongding Decoction could inhibit the signal transduction of p38MAPK signal transduction pathways,inhibit the activation of the microglia,and further more reduce the secretions of the inflammatory cytokines such as P substance,TNF-α and COX-2 in the L5-S2 medulla spinalis hence performing the actions of anti-inflammation and pain alleviation.