Studies On The Effect Of Li Dan Decoction On Gallbladder Motility And The Deterioration Mechanism Of Hyperuricemia Comorbid With Lschemic Stroke

Purpose:Gallstone disease is a kind of common digestive disease among worldwide,and cholecystectomy is the golden standard in treating gallstones.However,the recurance rate of gallstone disease is rather high due to the continuous sercretion of lithogenic bile.The Lidan Decoction has been clinically used in China in treating gallstones for many years and it showed a satisfied effect based on previous clinical observation.Nevertheless,the therapeutic mechanism of Lidan decoction is not clear.In present study,we established cholesterol gallstones model in guinea pigs to investigate the impact of Lidan Decoction on gallbladder notility and its protective effect on interstitial cajal-like cells of gallbladder.Meanwhile,we also applied TCMSP database to identify the potential active compounds of Lidan Decoction that related to its gallstone-preventing effect.In the second part of this study,we investigated in the pathological impact of hyperuricemia(HUA)on ischemic stroke.Up to date,the relations of hyperuricemia and cardiovascular diseases and metabolic syndrome are well established.However,it is still not clear whether hyperuricemia is a poor prognostic factor of stroke.Most of clinical studies showed hyperuricemia comorbid with stroke can increase the mortality and the chance of poor functional outcome,but other studies held an opposing view.In this study,we established a rat model of hyperuricemia comorbid with ischemic stroke to find out if hyperuricemia can serve as a potential risk factor of stroke,and its related mechanisms as well.Methods:Forty male guinea pigs were randomly divided into Control group,Model group,ursodeoxycholic acid group and Lidan Decoction group.Lithogenous diet was fed for eight weeks to introduce gallstone disease.At day 56,all animals were sacrificed and the following parameters were tested,including the prevalence rate of gallstone disease,and the number of cholesterol gallstones of each group,and the tonic contraction responses of gallbladder muscle strips to different concentrations of CCK-8 or Ach,and the density of interstitial cajal-like cells of gallbladder,and the activation of TNF-?/Caspase-8/Caspase-3 pathway,and the inflammatory cell infiltration in gallbladder.Meanwhile,we applied TCMSP database to screen the active compounds of Lidan Decoction that can potentially react with TNF-a/Caspase-8/Caspase-3 pathway.To identify the pathological changes of hyperuricemia(HUA)comorbid with ischemic stroke,the transient middle artery occlusion surgery(tMCAO)was applied to mimic ischemic stroke pathology,and the method of daily gavage of oxonic acid and uric acid was used to induce hyperuricemia.We defined the blood uric acid level exceeding 420 ? mol/L at day 7 as a successful model of hyperuricemia.Animals were equally divided into three groups:sham group,MCAO group and HUA+MCAO group.After 7 days HUA modeling,we applied tMCAO surgery on MCAO group and HUA+MCAO group,and sham operation was conducted on sham group.The survival rate and neuronal behavior test were recorded up to 7 days after MCAO surgery.The infract volume was measured by TTC staining,and the activation of NALP3 inflammasome pathway was detected by Western blot,and the density of nissl bodies was also measured by Nissl staining.Moreover,immunohistochemistry method were applied to detect the activation of microglia and NALP3 expression level.Results:Lidan Decoction showed preventive effect on gallstone disease by reducing the prevalence and decreasing the number of gallstones.After daily administrated Lidan Decoction,the tonic contractions of gallbladder muscle strips in response to CCK and Ach were significantly increased.The immunofluorescence result showed that Lidan Decoction exert anti-apoptosis effect on ICLCs of gallbladder,and the western blot and quantitative real-time PCR results showed Lidan Decoction can improve the mRNA expression of SCF/c-kit,and inhibite the cell apoptosis pathway TNF-?/Caspase 8/Caspase 3,and reduce the inflammatory cell infiltration as well.We identified in total 32 active compounds that can react with TNF-a/Caspase-8/Caspase-3 pathway,and 9 of them can be oral absorpt,and potentially exert their anti-apoptotic effect.In the second part of this study,HUA+MCAO model was successfully established.Comparing with MCAO group,HUA+MCAO group showed a lower rate of survival and poorer neurological function and greater infract volume as well.The western blot result showed HUA+MCAO group have a significant higher activation of NALP3 inflammasome at 24h and 48h after tMCAO and a greater production of IL-1?.Moreover,HUA+MCAO group showed less number of Nissl bodies and more activation of microglia in striatum region.Conclusion:Lidan Decoction can prevent apoptosis on ICLCs of gallbladder via inhibiting TNF-?/Caspase-8/Caspase-3 pathway,which can promote the contraction of gallbladder and then prevent gallstones formation.Further,several active compounds of LDT can act on the TNF-?/Caspase-8/Caspase-3 pathway through a multiple-targets pattern.In the HUA+MCAO rat model,HUA comorbid with ischemic stroke can lead to a higher mortality and poorer functional outcome,and the activation of NALP3 inflammasome followed by the relase of IL-1? resulted from the higher level of serum uric acid may involve in the exacerbation effect of HUA comorbidity.