| BackgroudsIrritable bowel syndrome(IBS)is one of the most common functional gastrointestinal disorders(FGDs),and diarrhea-predominant IBS(IBS-D)is the most common subtype among the IBS patients in our country.IBS is a multifactorial disease with complex pathophysiological mechanisms.Early life adverse(ELA)plays an important role in the occurrence of IBS,and visceral hypersensitivity(VH)is the core pathophysiological mechanism of the disease.The disease is prone to recurrent attacks possibly associated with the persistent visceral hypersensitivity,which has the negative influence on the patients and the increase the medical burden to the society.The epidemiological research indicates that ELAs,influencing the development of nervous and digestive systems,are positively related with the occurrence of IBS and/or the formation of VH.And the mechanism of the formation of VH mainly involves peripheral sensitization and central sensitization.And extracellular signal-regulated kinase(ERK)plays an important role in the formation of the VH by regulating synaptic plasticity.Continuous noxious stimulations increase the concentration of 5-HT,SP in the serum,colon and hippocampus.And the high level of 5-HT and/or SP can induce the influx of Ca2+,combined with the structure of CaMKII,and active the downstream factor ERK,inducing the expression of c-fos,and amplify sensory signals.Thus,ERK/c-fos signaling pathway plays an important role in the visceral hypersensitivity.Due to the recurrent attacks,the current treatment has not fully satisfied the patients requirements,so it is also one of the most important points to exploring safe and effective prevention and treatment methods is still the research focus of this disease.Traditional Chinese Medicine(TCM)treatment methods are effective to treat IBS-D,with holistic concept and treatment based on syndrome differentiation.Our team have been summarized that spleen-kidney Yang deficiency is the key syndrome of symptoms of the disease.Based on this,Wenshen-Jianpi method is established as the principle of the treatment and Wenshen-Jianpi prescription is established as the effective prescription.The clinical study has showed that Wenshen-Jianpi prescription can effectively ameliorate the symptoms of IBS-D,the total effective rate is 92.24%and decrease the recurrence rate in 6 months.However,the therapeutic mechanism remains unclear.Therefore,this study aims to explore the therapeutic mechanism of WenshenJianpi prescriptions via clinical and basic study,so as to provide new ideas for further understanding the mechanism of this decoction in the treatment of IBS-D,and to provide research basis for clinical transformation and application.Objectives1.To explore the correlation between early life adverse,the severity of IBS symptoms,visceral sensitivity index and the level of 5-HT,SP,Ca2+ in serum of IBS-D patients with spleen-kidney Yang deficiency.2.Based on ERK/c-fos signaling pathway,to explore the peripheral and central mechanism of Wenshen-Jianpi prescriptions on ameliorating visceral hypersensitivity in IBS-D rats with spleen-kidney Yang deficiency induced by maternal separation combined with gavage of senna solution.Methods1.Thirty-eight IBS-D patients with the syndrome of spleen-kidney Yang deficiency who visited the department of Gastroenterology and Endoscopy at Wangjing Hospital from November 2021 to January 2023 were selected,along with 10 control subjects.IBS-SSS,visceral sensitivity index scale and Childhood Trauma Questionnaire(CTQSF)were used to evaluate the severity of IBS symptoms,the degree of the visceral sensitivity and the experiences of early life adverse.The concentration of 5-HT,SP and Ca2+in serum were detected by ELISA.Spearman correlation analysis was used to explore the correlation between early life adverse,visceral sensitivity index,the severity of IBS symptoms and the concentration of 5-HT,SP and Ca2+in serum in IBSD patients with spleen-kidney Yang deficiency.2.The IBS-D model of spleen-kidney Yang deficiency was established by maternal separation combined with the gavage of senna solution.(1)The general condition and behavioral tests(Bristol stool form scale score and AWR score)were used to evaluate the severity of IBS symptoms.(2)The severity of spleen-kidney Yang deficiency was evaluated by detecting the concentration of cAMP and cGMP in serum by ELISA.(3)The levels of 5-HT,SP,pCaMKⅡ,pERK1,pERK2 and c-fos in colon tissues were detected by ELISA and/or Western Blot.Fecal microbiota was detected by 16S rRNA(in the previous study,we had detected the fecal microbiota and selected 5 important bacterial genera,and in this study,we only used the previous result to produce the correlation analysis).Fecal metabolites were detected by high-performance liquid chromatography-mass spectrometry.Spearman correlation analysis was used to explore the correlation among the level of pERK1,pERK2,c-fos,AWR score and the abundance of important bacterial genera and metabolites,respectively.(4)Western Blot was used to detect the levels of pERKl,pERK2 and c-fos in DRG tissue,and Spearman correlation analysis was used to explore the correlation between the expression of pERKl,pERK2,c-fos and AWR score.The expression levels of 5HT,SP,pCaMKⅡ,pERK1,pERK2 and c-fos in hippocampus tissue were detected by ELISA and/or Western Blot.Resting-state functional magnetic resonance imaging(fMRI)was used to evaluate brain neural activity.Spearman correlation analysis was used to explore the correlation between the levels of pERK1,pERK2,c-fos and AWR score and the data of neural activity signals,respectively.Results1.To explore the correlation between early life adverse,the severity of IBS symptoms,visceral sensitivity index and the level of 5-HT,SP,Ca2+in serum of IBS-D patients with spleen-kidney Yang deficiency(1)There were no statistically significant differences in general characteristics(gender,age,BMI)between the two groups(P>0.05).The IBS-SSS score for the control group was 22.62±23.59(score range 0-50),while the IBS-SSS score for the IBS-D with spleen-kidney Yang deficiency group(IBS-D group for short)was 201.50±41.78(score range 125-275).Compared with the control group,the VSI score(reverse score)of IBSD group was significantly decreased(P<0.0001),while there was no significant difference in the CTQ-SF total score between the two groups(P>0.05).The score for emotional abuse(EA)score was significantly increased(P<0.01),and the score for emotional neglect(EN)(reverse score)was significantly decreased(P<0.05)in IBS-D group compared to the control group.The correlation analysis among the CTQ-SF score(included the total score and subscale score),IBS-SSS score and VSI score showed that EA score had a weak negatively correlation with the VSI index(|R|=0.42,P=0.00022),while EA score(reverse score)had a weak positively correlation with the VSI index(|R|=0.30,P=0.0090).The correlation between the IBS-SSS and the CTQ-SF score was not yet significant(P>0.05).(2)Compared with the control group,the concertation of 5-HT and SP in the serum of IBS-D group were significantly increased(P<0.0001),and the concertation of Ca2+ in the serum of IBS-D group was slightly decreased,while there was no significant difference(P>0.05).The level of 5-HT and SP in the serum were negatively correlated weakly with CTQ-SF total score and EN score(reverse score)(|R|>0.3,P<0.05),and there was a positively correlation with EA score(reverse score)(|R|>0.3,P<0.05).PN score(reverse score)was negatively correlated with the concertation of 5-HT in the serum(|R|=0.32,P<0.05),and positively correlated with the concertation of Ca2+in the serum(|R|=0.38,P<0.01).the concertation of 5-HT and SP in the serum were positively correlated with IBS-SSS score(|R|>0.3,P<0.05),and negatively correlated with the VSI score(reverse score)(|R|>0.3,P<0.01).2.The peripheral-central effect mechanism of Wenshen-Jiangpi prescription intervening on visceral hypersensitivity of IBS-D rats with spleen-kidney Yang deficiency.(1)Behavioral performance:at the end of modeling,compared with control group,BSFS scores in the other groups were significantly increased(P<0.01),and AWR scores in the other groups at the pressure level of 40 mmHg,60 mmHg and 80 mmHg were significantly increased(40 mmHg:P≤0.05;60 mmHg:P<0.05;80 mmHg:P<0.01).At the end of the intervention,compared with the model group,BSFS scores in WenshenJianpi prescription(WJP for short)with medium concertation group and WJP with high concertation group were significantly decreased(P=0.0002,P<0.0001),and BSFS scores in the positive drug group were slightly decreased(P=0.28,P=0.27).Compared with the model group,at the pressure level of 40 mmHg and 60 mmHg,the AWR scores of the positive drug group,WJP with medium concertation group and WJP with high concertation group were significantly decreased(P<0.05),and at the pressure level of 80 mmHg,the AWR scores in other groups decreased slightly(P>0.05).(2)cAMP and cGMP levels in the serum.Compared with the control group,the concertation of cAMP and cGMP in the serum were significantly decreased in the model group(P<0.0001).Compared with the model group,the concertation of cAMP in the serum was significantly increased in the groups of WJP with medium concertation and WJP with high concertation(P<0.0001),the concertation of cGMP in the serum was significantly increased in the groups of positive drugs,WJP with medium concertation,WJP with high concertation and WJP with low concertation(P<0.0001).(3)Peripheral effects of Wenshen Jianpi prescription on visceral hypersensitivity in IBS-D rats with spleen-kidney Yang deficiency:Compared with the normal group,there was no significant difference in the concentration of 5-HT in the colon of the model group(P>0.05),the concentration of SP was significantly increased(P<0.0001),and the concentration of pCaMKⅡ was significantly decreased(P<0.0001).Compared with the control group,the relative expressions of pERKl,pERK2 and c-fos in the colon of the model group were significantly increased(P<0.01).Compared with the model group,the relative expressions of pERK1 and pERK2 in the groups of WJP with medium concentration and WJP with high concentration were significantly decreased(P<0.01),and the relative expressions of c-fos in the groups of positive drug,WJP with medium concentration and WJP with high concentration were significantly decreased(P<0.05).Spearman correlation analysis was used to analyze the correlation between AWR score and the relative expressions of pERKl,pERK2 and c-fos in colon tissue in each group at the end of intervention(12th week).The relative expressions of pERKl,pERK2 and c-fos were positively correlated with AWR score at the pressure level of 40 mmHg,60 mmHg,80mmHg(|R|>0.50,P<0.0001).There were 5 important bacterial genera,and the levels of pERK1、pERK2、cfos were positively correlated with the abundance of Bacteroides,Romboutsia Blautia,Clostridium(|R|>0.5,P<0.01),the level of c-fos was negatively correlated with the abundance of Candidatus_Saccharimonas(|R|>0.5,P<0.01).Fecal metabolites composition in the model group were different from that of control group,and WJP with medium concentration group were similar to that of control group.There are 593 different metabolites(DEMs)between the control group and model group,159 DEMs among them were upregulated and 434 DEMs among them were downregulated.There are 609 DEMs between the model group and WJP with medium concertation group,303 DEMs among them were upregulated and 306 DEMs among them were downregulated(FC≥1,VIP≥1,P<0.01).There are 393 DEMs grouped into the intersection,252 DEMs among them were upregulated by WJP,involved the KEGG pathways including steroid hormone biosynthesis,arginine and proline metabolism,neuroactive ligand-receptor interaction,and synaptic vesicle cycle.And 141 DEMs among them were downregulated by WJP,involved the KEGG pathway including amino acid biosynthesis,carbon metabolism,cocaine addiction and lipopolysaccharide biosynthesis.To select the key DEMs with the parameters of FC=2,VIP=1.5,P<0.01,and there were 13 DEMs strongly correlated with the relative expression of pERKl,pERK2 and c-fos in the colon tissue(|R|>0.7,P<0.01).(4)Central effects of Wenshen-Jianpi prescription on visceral hypersensitivity in IBSD rats with spleen-kidney Yang deficiency:Compared with the control group,the relative expressions of pERK1,pERK2 and c-fos in DRG tissues of the model group were significantly increased(P<0.01).Compared with the model group,the relative expressions of c-fos in the groups of positive drug and WJP with medium concentration were significantly decreased(P<0.05),and the relative expressions of pERKl and pERK2 were slightly decreased(P>0.05).the relative expression of pERK1,pERK2 and c-fos were weakly correlated with the AWR score at the pressure level of 40mmHg and 60mmHg(|R|>0.30,P<0.01).Compared with the control group,there was no significant different in the concertation of 5-HT in the hippocampus tissues in the model group(P>0.05),while the concentration of SP was significantly increased in the model group(P<0.01)and the concertation of pCaMK II was significantly decreased in the model group(P<0.01).Compared with the model group,there was no significant different in the concertation of 5-HT in the WJP with medium concertation group(P>0.05),while the concertation of SP was significantly decreased in the WJP with medium concertation group(P<0.05)and the concentration of pCaMK II were slightly increased in the WJP with medium concertation group(P)=0.061).Compared with the control group,the relative expressions of pERKl,pERK2 and c-fos in the hippocampus tissues in the model group were significantly increased(P≤0.0001).Compared with the model group,the relative expressions of pERK1,pERK2 and c-fos in the hippocampus tissues in the WJP with medium concentration group were significantly decreased(P<0.05).The results of resting-state functional magnetic resonance imaging:ALFF analysis showed that compared with the control group,neural activity in the dorsal hippocampal commission(dhc)and hippocampus(CA2,CA3)were reduced in the model group(P<0.005),and compared with the model group,the neural activity in the hippocampus(CA1,CA2)and lateral entorhinal cortex(LEnt)were reduced in the WJP with medium concentration group(P<0.005).fALFF analysis showed that compared with the control group,the neural activity in the dorsal hippocampal commission(DHC)were reduced in the model group(P<0.005),the neural activity in the periolfactory cortex(PRh)and LEnt were enhanced(P<0.005),and compared with the model group,the neural activity in the hippocampus(CA1),dentate gyrus(DG),dentate gyrus polymorpha(PoDG),outer olfactory cortex(Ect),PRh and LEnt were reduced(P<0.005).ReHo analysis showed that compared with the control group,the consistency of local neural activity in the hippocampus(CA2),PRh and LEnt were increased in the model group(P<0.005),and compared with the model group,the consistency of local neural activity in the hippocampus(CA2),PRh and LEnt were decreased in WJP with medium concentration group(P<0.005).The Spearman correlation analysis showed that the relative expressions of pERK1,pERK2,and c-fos were correlated with the AWR scores under pressure levels of 40mmHg,60mmHg,and 80mmHg(|R|>0.30,P<0.05).According to the difference of the neural activity between the model group and control group,the relative expressions of pERK1,pERK2 and c-fos in the hippocampus were negatively correlated with the neural activity in the dhc,hippocampus(CA2,CA3)and DHC(|R|≥0.59,P<0.05),and positively correlated with the neural activity in the hippocampus(CA2),PRh,LEnt(|R|≥0.65,P<0.01).According to the difference of the neural activity between the model group and WJP with medium concertation group,the relative expressions of pERK1,pERK2 and c-fos in the hippocampus were positively correlated with the neural activity in the hippocampus(CA1,CA2),LEnt,DG,PoDG,Ect,PRh(|R|≥0.65,P<0.01).Conclusions1.Early emotional abuse and emotional neglect experience during early life are positively correlated with the severity of IBS symptoms and visceral sensitivity index;The levels of 5-HT and SP in serum in IBS-D patients with spleen and kidney Yang deficiency are increased,and positively correlated with the severity of IBS symptoms,visceral sensitivity index,emotional abuse and emotional neglect experience in early life.2.Wenshen-Jianpi prescription can improve the symptoms and ameliorate visceral hypersensitivity of IBS-D model rats with spleen-kidney Yang deficiency:(1)peripheral effect:Wenshen-Jianpi prescription can down-regulating the expression of pERK1,pERK2 and c-fos in the colon and regulating the related bacteria genera and metabolites.(2)central effect:Wenshen-Jianpi prescription can down-regulating the expression of pERK1,pERK2 and c-fos in the hippocampus and regulating the neural activity in the hippocampus. |
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