| Hemorrhoids is a common benign anorectal disease.Milligan-Morgan is still the first choice for patients with grade Ⅲ-Ⅳ hemorrhoids,and pain is an important reason for patients to refuse or fear surgery.There are two main causes of postoperative wound pain in mixed hemorrhoids:surgical trauma itself and anal sphincter spasm.Widely used in clinical and animal experiments,the "Guang Tong Xiao foam aerosol" has been proved to have good effects on spasmodic pain relief,anti-inflammatory,detumescence,and hemostasis.Previous pharmacological studies showed that the "Guang Tong Xiao foam aerosol" may reduce the excitability of DRG neurons by inhibiting the production of PGE2 and the expression of NR2B in spinal dorsal horn neurons.At the same time,it can reduce the expression of TNF-α,IL-6,IL-1β and iNOS in perianal tissues of rats,so as to play the role of anti-inflammatory and analgesic.Guang Tong Xiao foam aerosol can also significantly relieve spasm of anal sphincter.The mechanism may be that its effective components THP and Pae can block voltage gated calcium channels,reduce calcium influx and release of calcium,thereby inhibiting the excitation contraction coupling of smooth muscle.The research contents of this thesis are divided into four parts.First,the clinical randomized controlled method is applied to evaluate the efficacy of Guang Tong Xiao foam aerosol after hemorrhoid surgery;UPLC-Q-TOF-MS was used to analyze the metabolites of the main active components of Guang Tong Xiao foam aerosol in rat bile;The influence of active ingredients of Guang Tong Xiao foam aerosol on smooth muscle cell activity was detected by an in vitro inflammatory model;The effects of effective components of Guang Tong Xiao foam aerosol on ion channels of smooth muscle cells were detected by automatic patch clamp,high throughput ion channel determination.Part oneClinical study of the effect of Guang Tong Xiao foam aerosol on postope-rative anal pain in patients with mixed hemorrhoidsObjective A randomized controlled trial was conducted to observe the efficacy of Guang Tong Xiao foam aerosol,compound polymyxin B ointment and vaseline ointment on the complications of external hemorrhoids after external hemorrhoids ligation,so as to evaluate the analgesic,detumescence and healing effects of Guang Tong Xiao foam aerosol,and to evaluate the safety of Guang Tong Xiao foam aerosol,so as to provide a reliable basis for its new drug research.Methods In this study,90 patients with mixed hemorrhoids were selected and screened according to the diagnostic,inclusion and exclusion criteria.They were randomly divided into treatment group(Guang Tong Xiao foam aerosol),control group(compound polymyxin B ointment)and blank group(Vaseline)according to 1:1:1 ratio.to evaluate the pain,edema,urinary retention,analgesic drugs and wound healing after external hemorrhoids ligation.Results The analgesic effect of Guang Tong Xiao foam aerosol is better than that of control group and blank group.The results showed that the analgesic effect of Guang Tong Xiao foam aerosol in the first three days after operation was better than that in the control group and blank group,and the analgesic effect on the seventh day after operation was better than that in the blank group;The pain relieving effect of Guang Tong Xiao foam aerosol was better than that of the control group and blank group second,third,seventh days after operation,and the analgesic effect of fourteenth days after operation was better than that of the blank group;At the same time,the application of pain relieving drugs in the group of postoperative pain was better than that in the control group and blank group;In terms of preventing edema,the performance of Guang Tong Xiao foam aerosol was better than that of the control group and blank group on the second,fifth day after operation;In terms of wound healing,the healing rate of Guang Tong Xiao foam aerosol was better than that of the control group and blank group third weeks after operation.Conclusion Guang Tong Xiao foam aerosol can safely and effectively relieve wound pain after external ligation,prevent postoperative edema and promote wound healing.Part twoUPLC-Q-TOF-MS analysis of metabolites of the Guang Tong Xiao foam aerosol main active components of rat’s bileObjective The metabolites of eight main active components in bile of rats after rectal administration of Guangtongxiao were determined.Methods After the rectum was given to Guang Tong Xiao foam aerosol,bile samples were collected from the bile duct and continued for 5h after administration.Blank bile was collected before administration.The metabolites of BBR,DHC,THP,Corydalin,Magnolflorin,MAG,Obacunone,Albiflorin were determined by UPLC-Q-TOF-MS.Results The chemical constituents of the active part of Guangtongxiao were analyzed and identified by positive and negative modes,and the representative chromatograms of the two modes were obtained.Eight peaks were identified by comparison with the standard compounds BBR,DHC,THP,Corydalin,Magnolflorin,MAG,Obacunone,Albiflorin.Conclusion UPLC-Q-TOF-MS method was established to identify the metabolites of Guangtongxiao in rat bile samples collected 5 hours after rectal administration.A total of 8 main active components and 60 metabolites were detected,of which 21 were reported for the first time.We have a better understanding of the metabolic process in vivo after rectal administration of Guangtongxiao.These results lay a foundation for further study on the pharmacology and pharmacokinetics of Guangtongxiao,and help to clarify the mechanism of action of Guangtongxiao.Part threeEffects of THP and Pae paeoniflorin on the activity of smooth muscle cellsObjective Observe the effects of THP and Pae on the activity of rat colonic smooth muscle cells.Methods Objective to isolate,purify,culture and identify rat colonic smooth muscle cells,observe the effect of different concentrations of THP and PAE on the viability of smooth muscle cells,and determine the safe range.The effects of the two drugs on the secretion of no by rat colonic smooth cells were detected by Griess reagent.The concentrations of inflammatory cytokines(TNF-α,IL-1β,IL-6,VEGF,PGE2)were detected by ELISA kit.Results THP and PAE could significantly decrease the level of no in a concentration dependent manner.THP and PAE can inhibit IL-6,PGE2,enhance the inhibition and protection of inflammatory response.Conclusion THP and PAE,the main components of Guangtongxiao,may release IL-6,TNF-α,PGE2,NO and other inflammatory mediators from peripheral non nerve cells,so as to reduce peripheral sensitization and central sensitization and relieve postoperative wound pain.Part fourEffects of tetrahydropalmatine and paeoniflorin on ion channels of rat anal sphincter cells.Objective observe the effects of THP and PAE on the ion channels of rat anal sphincter cells,and explore the relationship between the effective components of Guang Tong Xiao and the calcium channels of smooth muscle cells,and further elucidate the mechanism of spasmodic effect of Guang Tong Xiao foam aerosol.Methods The effects of the effective components of Guang Tong Xiao foam aerosol on the concentration of intracellular free calcium in rat anal sphincter muscle cells,the effects of sodium potassium pump and calcium ion pump activity and electrophysiology were detected by automatic patch clamp,high throughput ion channel determination.Results The stimulating effect of THP and PAE on ICa-L current leads to the inhibition of ICa-L current,but the inhibition is weak;THP and PAE groups had significant effects on calcium influx of anal sphincter cells;PAE inhibited the process of extracellular potassium ion entering into cells;THP,PAE and mixed drug groups inhibited Ca2+-ATPase in the culture supernatant of rat anal sphincter cells;Conclusion THP and PAE can inhibit the abnormal increase of ICa-L in rat anal sphincter cells and raise the current voltage curve,which indicates that THP and PAE can inhibit the contraction of anal sphincter cells.At the same time,high-throughput ion channel analyzer showed that PAE could significantly inhibit the process of extracellular potassium ion entering into cells,so as to reduce the excitability of smooth muscle cells.THP,PAE and mixed drug groups inhibited Ca2+-ATPase in the culture supernatant of rat anal sphincter cells.It can affect Na+-Ca2+exchange,inhibit the excitation contraction coupling of rat anal sphincter cells,and reduce the excitability of smooth muscle cells. |
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