| Research BackgroundRectal prolapse(RP)is a rare benign disease in anorectology,which is called"prolapse of the anus" in ancient Chinese medicine.It has the advantages of good efficacy,simple operation,and low pain.Xiaozhiling is the representative drug of the fixation agent.Our research team has long used the injection of Xiaozhiling to treat rectal prolapse and obtained good efficacy.However,there is a certain recurrence rate after Xiaozhiling injection,which has become a difficult point in treatment.To address the problem of recurrence,our group had exploratively improved the double-layer four-step injection(abbreviated as"double-layer four-step")to eight-step injection in the perirectal space(abbreviated as"single-layer eight-step"),after the observation of the preliminary case series,it was found that the single-layer eight-step injection of elimination hemorrhoids could reduce recurrence to some extent.The results lacked evidence-based medical evidence because a comparative study with a two-layer four-step injection was not conducted.At the same time,improving injection methods to reduce recurrence alone is not enough to solve the problem.According to our experience,many factors affect the recurrence of rectal prolapse treated by Xiaozhiling injection,but there is no quantitative tool to accurately predict the recurrence.Therefore,it is necessary to continue indepth research and find out various related risk factors that affect recurrence,establish a clinical prediction model,and quantify the probability of recurrence,to carry out key interventions in advance for the high recurrence population and formulate individualized treatment plans.As one of the possible factors affecting recurrence,the injection method needs to be discussed in-depth,that is,to compare the short-term and long-term effects of single-layer eight-step and double-layer four-step injection,and to further analyze the impact of different injection methods on the cure rate and recurrence.To provide evidence-based medicine.Compared with clinical studies,the basic research on the treatment of rectal prolapse with Xiaozhiling injection is still relatively backward,and its mechanism of action has not been elucidated so far.As an important tool in TCM research,integrative pharmacology has resolved the molecular biological network and pharmacological effects of TCM compound from molecular,cellular,and tissue levels.By applying the research methods of integrative pharmacology to elucidate the mechanism of action of Xiaozhiling injection in the treatment of rectal prolapse and exploring its key targets and pathways of action,it not only promotes the development of basic research but also has important guiding significance for clinical research.Research objectives1.To investigate the factors influencing the recurrence of rectal prolapse after the administration of Xiaozhiling injection,to establish a clinical prediction model,to objectively assess the probability of recurrence,to focus on strengthening timely interventions for people with high recurrence,and to provide individualized diagnosis and treatment plans to reduce recurrence.2.A retrospective cohort study was conducted using a real-world research method to balance the confounding factors between groups using propensity score matching method,and to compare the near and long-term efficacy of two injection methods of Xiaozhiling single-layer eight-step and double-layer four-step,and to provide evidence-based medicine for clinical research.3.We used an integrative pharmacological research method to explore the potential mechanism of action of the injections of Xiaozhiling in the treatment of rectal prolapse,providing the theoretical guide for basic and clinical research.Research methods1.Construction of a prediction model for the recurrence of rectal prolapse treated with Xiaozhiling injectionPatients with rectal prolapse who underwent Xiaozhiling injection in the Department of Anorectal Medicine,Guang'anmen Hospital,Chinese Academy of Traditional Chinese Medicine were selected from January 1,2009,to December 31,2019,and 150 cases were finally enrolled according to the diagnosis and inclusion and exclusion criteria.Ten potentially influential factors,including age,sex,body mass index,disease duration,prolapse length,prolapse shape,classification and grading,anal sphincter function,whether combined with small bowel prolapse,injection method,and follow-up data at 3 months,6 months,1 year,2 years,3 years,5 years,and 10 years after surgery were collected.IBM SPSS 26.0 statistical software was used to describe the basic information of the entire cohort of 150 patients and to provide a general description of the characteristics of the patients in the recurrence and nonrecurrence groups.Kaplan-Meier survival curves for the whole cohort were drawed using GraphPad Prism 8.0 and survival analysis was performed.Two methods were used to screen influential factors as covariates,the Cox univariate and multifactor methods,and the randomized survival forest method to identify risk factors associated with recurrence and perform a Cox proportional risk assumption test,where categorical variables were plotted using the survminer package of the R3.6.3 software for Kaplan-Meier survival curves between two groups,and the survivor package for statistical analysis of survival data.Differences between groups were compared by Log-rank test,and P<0.05 was considered to demonstrate statistically significant differences.The risk assumption testwas satisfied if there was no crossover of the curves.For continuous variables among the influencing factors,the Schoenfeld residual method was used to test for no linear correlation,and the risk assumption test was satisfied.Based on the recurrence influencing factors screened by the above two methods,prediction models were established separately,and the difference in their ability to predict recurrence was evaluated and compared by the area under the time-dependent ROC curve,and the model with better prediction ability was selected.Further,the foreign package?survival package and rms package of R3.6.3 software were used to draw nomogram for visual presentation of the prediction models,and The calibration degree of the model is checked by the calibration curve.2.Comparison of the efficacy of single-layer eight-step and double-layer four-step injection of Xiaozhiling in the treatment of rectal prolapsePatients with rectal prolapse who underwent Xiaozhiling injection at the Department of Anorectal Department,Guang'anmen Hospital,Chinese Academy of Traditional Chinese Medicine between January 1,2009,and December 31,2019,were selected,and 150 cases were finally enrolled according to the diagnosis an-d inclusion and exclusion criteria.Patients'age,gender,body mass index,disease d-uration,prolapse length,prolapse shape,classification and grading,anal sphincter fu-nction,whether combined with small bowel prolapse,total preoperative symptom score,concentration ratio(1:1,2:1)and dosage of Xiaozhiling,and the total dos-age of undiluted Xiaozhiling was calculated.Postoperative complications(urinar-y retention,constipation,diarrhea)and adverse events(abdominal cramps,anal cra-mps,fever,anal bleeding,rectal mucosal ulceration and necrosis,prostatitis)were re-corded.Follow-up data were collected at 3 months,6 months,l year,2 years,3 y-ears,5 years,and 10 years postoperatively.The cohort was divided into two grou-ps:single-tier eight-step and double-tier four-step.The nearest neighbor matching method of propensity score matching(PSM)was used to balance the confounding factors between the two groups,and the caliper value was set at 0.2.PSM analysis was implemented by the PSM extension procedure of IBM SPSS 24.0 statistical software.The final cases were compared after 1:1 matching.Among the recent efficacy indicators,the primary comparison was the cure rate and total effective rate between the two groups,comparing the total symptom scores on postoperative days 1,3,7,and 15.The occurrence,severity,management,and regression of complications and adverse events were observed secondarily.Among the long-term efficacy indicators,the total recurrence rate,the accumulative recurrence-free survival rate,and therefore the median survival time without recurrence were chiefly compared between the 2 groups.The length of prolapse and concomitant symptoms after recurrence and treatment after recurrence were also observed.IBM SPSS 26.0 statistical software was used for data analysis,and all statistical analyses were performed using a two-sided hypothesis test with a test level of a=0.05,and the level of significance was P<0.05.The measurement data conformed to a normal distribution and were expressed using the standard deviation of the mean,and the t-test for independent samples was used for comparison between groups;for those with non-normal distribution,the median(interquartile spacing)was used,and the Wilcoxon ranksumtest was used for comparison between groups.The composition ratios of the count datawere expressed using frequency(percentages).Comparisons of the composition ratios of categorical data were made using the chi-square test or Fisher's exact probability method.The Wilcoxon rank-sum test was used for comparison of rank data.Violin plots of prolapse length before and after PSM were drawn using GraphPad Prism 8.0.Survival curves of the two groups were plottedusing the survminer package of R3.6.3 software,and the survival package was used for statistical analysis of survival data and Log-rank test was used to compare the differences between groups,and differences were considered to be significant at P<0.05.Test efficacy was calculated using PASS 15.0.1 software.3.Based on integrated pharmacology to explore the mechanism of action of Xiaozhiling injection for rectal prolapseBased on the integrated pharmacology platform(TCMIP V2.0),we used the integrated pharmacology research method to predict the drug and disease targets,and then constructed the core target network for the treatment of rectal prolapse with Xiaozhiling,performed GO function analysis and KEGG pathway enrichment analysis on the screened key targets,and finally established a multidimensional network diagram of "drug-chemical component-core target-key pathway-disease",which was established to reveal its potential mechanism of action.Research results1.Construction of a prediction model for the recurrence of rectal prolapse treated with Xiaozhiling injection1.1 Follow-up results and overall survival analysisDuring the follow-up,4 cases died and 35 cases were lost to follow-up(the cumulative number of cases lost to follow-up after the second and third follow-up).There were 79 recurrences(52.7%)and 71 non-recurrence(47.3%).150 patients had a median survival time without recurrence of 60 months(95%CI(46.5-73.5)).The cumulative recurrence-free survival rates at 3 months,6 months,1 year,2 years,3 years,5 years,and 10 years after surgery were 93.3%,87.3%,80.3%,68.2%,55.1%,38.9%,and 22.1%,respectively.1.2 Analysis of clinical baseline characteristics of the entire cohortThe age of the registered patients was skewed with a median age of forty se-ven years(28.75-68.25years).There were eighty one males and sixty nine females,w-ith a male-to-female magnitude relation of 1.17:1.The body mass index was 23.66±3.54.57.3%of the cases were less than 10 years old,40%were 10-50 year-s old,and 2.7%were more than 50 years old.The length of prolapse was less than 6 cm in 62.7%of cases.Cylindrical prolapse was the most common shape,accounting for 64.7%.The proportion of type ? and degree ? and type ? and d-egree ? prolapse was higher(70%).About half of the patients had a weakened anal sphincter,and 16%of patients had no contraction of the anal sphincter.T-he proportion of patients with concomitant small bowel prolapse was slightly h-igher at 52%.The proportion of low dose and medium to a high dose of the undiluted Xiaozhiling was about half each.A single eight-step injection was perf-ormed in 84 cases and a double four-step injection in 66 cases.1.3 Comparison of clinical baseline characteristics of patients in the relapsed group and the non-relapsed groupCompared with the non-recurrence group,the recurrence group had a majority of elderly patients,a majority of patients with combined small bowel prolapse,and a greater percentage of patients with double-layer four-step injection,with a statistically significant difference(P<0.01).The majority of patients in the relapse group had a disease duration of more than 10 years,and the difference was statistically significant(P<0.05).The remaining two groups had no statistically significant differences in gender,body mass index,prolapse length,prolapse shape,fractional grading,anal sphincter function,and female patients who delivered versus those who did not deliver(P>0.05).1.4 Screening of risk factors and establishment of the recurrence prediction model1.4.1 Cox univariate and multifactorial approachThe ending variable was whether recurrence after injection and the influencing factors with P?0.1 in the univariate analysis were included in the Cox multifactor analysis using the input method,and the risk factors affecting recurrence were screened and conformed to the Cox proportional risk assumption test,and finally as follows:type ? and degree ? of the fractional division(X1,P=0.000<0.01),anal sphincter weakness(X2,P=0.032<0.05),small bowel prolapse(X3,P=0.018<0.05),and injection method(single-layer eight-step,X4,P=0.016<0.05).The formula of the prediction model developed was:h(t,X),h0(t)exp(1.344*X1+0.730*X2+0.610*X3-0.592*X4).1.4.2 Randomized survival forest approachThe ending variable was whether recurrence occurred after injection,and t-he risk factors affecting recurrence were screened and conformed to the Cox proportional risk assumption test and ended up as follows:type ? and degree? of the fractional division(X1,?=0.763,P=0.069>0.05),type ? and degree? of the fractional division(X1,?=1.278,P=0.001<0.01),and reduced anal sp-hincter function(X2,?=0.558,P=0.054>0.05),anal sphincter weakness(X2,?=0.703,P=0.037<0.05),small bowel prolapse(X3,P=0.010<0.05),prolapse length(X4,P=0.158>0.05),age(X5,P=0.027<0.05),and injection method(single-layer eight-step,X6,P=0.009<0.05).The formula of the prediction model developed was:h(t,X)=h0(t)exp(0.763/1.278*X1+0.558/0.703*X2+0.648*X3+0.058*X4+0.012*X5-0.675*X6).1.5 Performance evaluation of two recurrence prediction modelsAccording to the area under the Time-dependent ROC curve,AUC(t)showed that the Cox prediction model built from six risk factors screened by randomized survival forest had higher AUC values at each postoperative follow-up node and its mean value was greater than the former(0.803 versus 0.773)compared with the Cox single-factor versus multi-factor screening method(4 risk factors).1.6 Plotting the nomogram prediction modelNomogram were drawn to visually present the prediction model built from risk factors screened by the randomized survival forest approach.1.7 Calibration curves to evaluate the calibration of the nomogram prediction modelAs shown by the calibration curves,the predicted recurrence probabilities of the column line plot prediction model at 3 months,6 months,1 year,2 ye-ars,3 years,5 years,and 10 years after injection were generally consistent with the actual recurrence probabilities.2.Comparison of the efficacy of single-layer eight-step and double-layer four-step injection of Xiaozhiling in the treatment of rectal prolapse2.1 Baseline information before PSM in the single-layer eight-step group and double-layer four-step groupBefore PSM,there were no statistically significant differences in gender,age,body mass index,disease duration,classification and grading,anal sphincter function,prolapse shape,small bowel prolapse,and total preoperative symptom score between the two groups(P>0.05),but the differences in prolapse length between the 2 groups were considered significant(P<0.05).The prolapse length as an independent risk factor needs to be taken as a PSM method to balance this difference between the two groups.2.2 Baseline information after PSM in the single-layer eight-step group versus the double-layer four-step groupAfter PSM,35 cases failed to match in the single-layer eight-step group and 17 cases failed to match in the double-layer four-step group,both of which were excluded.49 cases were finally included in both groups,which were balanced and comparable between the groups.2.3 Comparison of recent outcomes after PSM between the single-layer eight-step group and the double-layer four-step groupThe healing rate of the single-layer eight-step group was higher than that of the double-layer four-step group,and also the distinction was statistically signifi-cant(P<0.05),and also the difference in the total effective rate between the 2 gro-ups wasn't statistically significant(P>0.05).In the comparison of the total sympto-m scores of the two groups on the 1st,3rd,7th,and 15th days after injection,the difference wasn't statistically significant on the first day after surgery(P>0.05),and on the third,seventh and fifteenth days after surgery,the total symptom scores of the single-layer eight-step group were lower than those of the double-layer f-our-step group,and the prolapse symptoms improved better,and the difference w-as accepted as indicative of significant differences(P<0.05).The occurrence grade of complications was mild in both groups.A total of 3 cases(6.1%)occurred in the single-layer eight-step group,including 1 case of urinary retention and 1 case of constipation,and no diarrhea occurred.A to-tal of 6 cases(12.2%)occurred in the double-layer four-step group,including 2 cases of urinary retention,3 cases of constipation,and 1 case of diarrhea.T-he differences between the two groups were not considered significant(P>0.05).The adverse events that occurred in both groups were mild,with a differen-ce in the overall incidence(P<0.05).A total of 14 cases(28.6%)occurred in the single-layer eight-step group,including 8 cases of small abdominal cramps,3 cases of anal cramps,1 case of fever,1 case of anal bleeding,1 case of decre-ased blood pressure and heart rate,and no rectal mucosal ulceration,necrosis or prostatitis occurred.A total of 6 cases(16.3%)occurred in the double-layer four-step group,including 1 case of small abdominal cramps,1 case of anal cra-mps,1 case of fever,2 cases of anal bleeding,and 1 case of rectal mucosal ulc-ers,without the occurrence of prostatitis and decrease in blood pressure and h-eart rate.Among them,the small abdominal cramps occurred significantly morei-n the single-layer eight-step group than in the double-layer four-step group,an-d the difference was statistically significant(P<0.05).The remaining two grou-ps had no statistically significant differences in the occurrence of anal cramps,fever,anal bleeding,rectal mucosal ulceration and necrosis,prostatitis,and decre-ase in blood pressure and heart rate(P>0.05).One case of an unexplained decr-ease in blood pressure and heart rate occurred in the single-layer eight-step gr-oup,which returned to nor-mal after active treat-ment and did not leave any se-quelae.One case of columnar necrosis of the intestinal wall forming a sinus tra-ct after blood in the stool occurred in the dou-ble-layer four-step group,whic-h was discharged after 1 month of conservative treatment and improved.2.4 Comparison of long-term outcome after PSM between the single-layer eight-step group and the double-layer four-step groupThe overall recurrence rate in the single-layer eight-step group was 51.0%(25/49).The median survival time without recurrence was 60 months(95%CI(45.2-74.8)).The cumulative recurrence-free survival rates at 3 months,6 m-onths,1 year,2 years,3 years,and 5 years after surgery were 95.9%,91.8%,87.6%,73.4%,58.2%,and 31.0%,respectively.The overall recurrence rate was 65.3%(32/49)in the two-tier four-step group.The median recurrence-free surviva-1 time was 24 mont-hs(95%CI(11.4-36.6).The cumulative recurrence-free surv-ival rates were 87.8%,79.6%,66.7%,49.4%,38.4%,and 25.8%at 3 months,6 months,1 year,2 years,3 years,and 5 years after surgery,respectively.There was no difference in the total recurrence rate between the two groups(P=0.152).The cumulative recurrence-free survival rate was higher in the single-layer eight-step group than in the double-layer four-step group,and the distinction w-as statistically significant(P<0.05).And median survival time without recurrenc-e was longer(60 mo.vs.24 mo.).There were 25 cases of recurrence in the single-layer eight-step group,including thirteen males and twelve females,the recentest was eighty nine years old,the youngest was fifteen years old,ten cases were over sixty years old,thirteen cases were over ten years old,and 16 cases were combined with small bowel prolapse.There were 32 cases of recurrence in the double-layer four-step group,including sixteen males and sixteen females,the oldest being eighty nine years old,the youngest seventeen years old,13 cases over 60 years old,18 cases with a disease duration of 10 years or more,and 16 cases of combined small bowel prolapse.When comparing the length of prolapse in the two groups of recurrent patients,the single-layer eight-step group was shorter than the double-layer four-step group,and the distinction was statistically significant(P<0.05).The differences in anal swelling,anal incontinence,and constipation occurring in both groups were not statistically significant(P>0.05).Thirteen cases in the single-layer eight-step group continued to come to our hospital after recurrence,among which eight cases underwent single-layer eight-step injection again,one procedure for prolapse and hemorrhoids,one open suspension of rectal prolapse,one laparoscopic rectal prolapse suspension,one partial resection of transanorectal sigmoid colon,and one anal circumcision combined with external sphincter folding.In the double-layer four-step group,11 cases continued to come to our hospital after recurrence,among which 1 case underwent double-layer four-step injection again,4 cases underwent single-layer eight-step injection,1 case underwent the procedure for prolapse and hemorrhoids,l case underwent open suspension of rectal prolapse,2 cases underwent laparoscopic rectal prolapse suspension.l case underwent a single-layer eight-step injection combined with automatic hemorrhoid ligation.1 case recurred after the second single-layer eight-step injection and underwent laparoscopic rectal prolapse suspension for the third time.3.Based on integrated pharmacology to explore the mechanism of action of Xiaozhiling injection for rectal prolapseThrough the integrated pharmacology platform(TCMIP V2.0),a drug similarity threshold of?0.6 was set,and after removing the duplicate targets,a total of 304 drug targets of tannic acid,the active ingredients of Xiaozhiling,and 55 targets of rectal prolapse were obtained,and a core target network was constructed to screen out 6 key targets of drug action(NFKB2,IKBKB,CHUK,NFKB1,NFKBIA,AKT1).The biological processes involved include cell cycle arrest,positive regulation of transcription,DNA-templated,protein phosphory-lation,positive regulation of transcription by RNA polymerase ?,signal transd-uction,and cellular response to tumor necrosis factor,etc.KEGG pathway enr-ichment analysis showed that most of the signaling pathways were related to NF-?B signaling pathway,namely RIP-mediated NF-?B activation via ZBP1,TR-AF6 mediated NF-?B activation,TAK1 activates NF-?B by phosphorylation an-d activation of IKKs complex,NF-?B is activated and signals survival,Activat-ion of NF-kappaB in B cells,and FCERI mediated NF-?B activation.Study conclusions1.Construction of a prediction model for the recurrence of rectal prolapse treated with Xiaozhiling injectionTo screen the factors influencing recurrence of rectal prolapse treated with the elimination of Xiaozhiling injection and to establish a clinical prediction model.Using whether recurrence was the outcome variable and considering the relationship between time without recurrence and recurrence outcome,two methods:Cox single-factor and multi-factor methods and randomized survival forest method were used to screen recurrence influencing factors and establish prediction models,and the Cox proportional risk model built by the latter had a better predictive performance for recurrence.The nomogram prediction model of the probability of recurrence at 3 months,6 months,1 year,2 years,3 years,5 years,and 10 years after injection was further constructed for visual presentation.In which higher scores of age,staging score,anal sphincter function,prolapse length,and small bowel prolapse were associated with a higher probability of postoperative recurrence.The probability of recurrence using a single-layer,eight-step injection was lower than that of a double-layer,four-step injection.Validation using calibration curves showed that the nomogram predictionmodel predicted the likelihood of recurrence generally agree-ment with the actual likelihood of recurrence.Future prospective studies with expanded sample size and inclusion of more influencing factors(e.g.,imaging,omics,etc.)are needed to further improve the initially established prediction model and validate it using external cohort data for practical application in the clinic to improve physicians' clinical decision-making ability.2.Comparison of the efficacy of single-layer eight-step and double-layer four-step injection of Xiaozhiling in the treatment of rectal prolapseThe single-layer eight-step group had a higher near-term cure rate than the double-layer four-step group,and the total symptom score on postoperative days 3,7,and 15 was lower than the latter,with a better improvement of prolapse symptoms.The near-term overall effective rate was comparable in both groups,and no serious complications or adverse events occurred in either group.In the long-term outcome,there was no difference in the total recurrence rate between the two groups.The single-layer eight-step group had a higher cumulative recurrence-free survival rate,a longer median survival time without recurrence,and a shorter prolapse length in patients with recurrence than the double-layer four-step group.The single-layer eight-step injection can be recommended as the procedure of choice for injection therapy.3.Based on integrated pharmacology to explore the mechanism of action of Xiaozhiling injection for rectal prolapseThrough integrated pharmacological study,the key targets of action for obtaining the elimination of Xiaozhiling injection for rectal prolapse are NFKB2,I-KBKB,CHUK,NFKB1,NFKBIA,AKT1,and among the involved signaling pathways,six NF-?B signaling pathways were associated,and it was hypothesized that the activation of NF-?B signaling pathway was an important link for abscess in injection to produce its effect.This laid a certain theoretical foundation for future in vivo and ex vivo experimental validation and also has important guiding significance for clinical research. |
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