Interventional Effect Of Dahuang Mudan Decoction In A Rat Model Of Acute Pancreatitis

Acute pancreatitis(AP)is a common critical disease of the digestive system.The incidence rate is increasing year by year,and various serious complications are prone to occur.The mortality rate of severe patients remains high,which is a serious challenge to clinical medicine.Traditional Chinese medicine has unique advantages in clinical treatment of AP.According to the main pathogenesis of damp-heat stasis stagnation and obstruction of qi in the viscera,the principles of clearing the viscera and releasing turbidity,promoting blood circulation and dissipating blood stasis,promoting qi and relieving pain are clarified.The application of traditional Chinese medicine can control the complications and reduce the abdominal pain and other symptoms.Dahuang Mudan Decoction(DMD)is a classic prescription of traditional Chinese medicine,which has long been effective in the clinical treatment of AP,but the specific mechanism of its effective intervention is not clear.It has clear practical significance and value to solve the problem of unclear mechanism of action and therapeutic targets of DMD.This study aimed to investigate the expression changes of key molecules of HMGB1/TLR4/NF-κB and HMGB1/RAGE/AKT signaling pathways in rat AP model,and the effect of DMD on the expression of effector molecules in the pathway,from "inflammation" and From the perspective of "apoptosis",the mechanism of DMD’s effective intervention in AP was revealed,and the following results were obtained:1.The rat AP model was reconstructed by retrograde injection of sodium taurocholate into the pancreatic duct.The survival status of the rats in the model group was observed,the serum biochemical indexes were detected,and the pancreatic pathological tissue sections were observed to evaluate the reconstruction of the model.It was found that compared with the blank group,the rats in the model group generally had poorer survival status after modeling,showing symptoms such as squinting,messy coat,drowsiness and sleepiness;serum amylase(AMS),alanine aminotransferase(Alanine)The contents of transaminase(ALT),aspartate transaminase(AST),and Creaction protein(CRP)were significantly increased,and the content of cholinesterase(Ch E)was significantly decreased(P<0.05).Histological observation showed that the pancreatic acinar cells in the model group were massively necrotic,the lobular duct structure was blurred,the interlobular septum was significantly widened,and a large number of congestion,edema and inflammatory cell infiltration were seen.The results showed that the rat AP disease model was successfully reconstructed.2.By detecting serum biochemical indexes,observing the histopathological and ultrastructural changes of rat pancreas,and analyzing the overall effect of DMD on AP in rats.It was found that compared with the model group,the contents of AMS,ALT,AST and CRP in each administration group decreased,while the content of Ch E increased,especially in the DMD high-dose group(P<0.05).There were different degrees of acinar cell necrosis in the pancreas of the rats in each administration group,the structure of the lobular ducts was blurred,the interlobular septum was widened,and hemorrhage and inflammatory cell infiltration were seen,but they were all relieved to varying degrees compared with the model group,especially with DMD high-dose group.The changes in the shape of nuclear membrane and structural defects of the acinar cells in each administration group,the scattered rough endoplasmic reticulum structure,and the swelling and transparency of mitochondria were improved to varying degrees,especially in the DMD high-dose group.It shows that DMD can regulate the contents of serum AMS,ALT,AST,Ch E and CRP,improve the damage of pancreatic tissue,and have a positive effect on the course of AP.3.Real-time quantitative polymerase chain reaction,Western blotting,and immunohistochemistry were used to detect the protein expression and phosphorylation level of key molecules in the HMGB1/TLR4/NF-κB signaling pathway,respectively,and the enzyme-linked immunosorbent assay was used to detect Contents of related inflammatory factors in pancreatic tissue.It was found that compared with the blank group,high mobility group protein B1(HMGB1),Toll-like receptor 4(TLR4),and Myeloid differentiation factor 88(Myeloid differentiation factor 88)in the pancreatic tissue of the model group were significantly increased.factor 88,MYD88),interleukin-1 receptor-associated kinase 2(IRAK2),interleukin-1 receptor-associated kinase 4(IRAK4),nuclear factor κB(Nuclear factor kappa-B,NF-κB)gene protein expression and related protein phosphorylation levels were significantly increased(P<0.05);,IRAK2,IRAK4,NF-κB gene protein expression and related protein phosphorylation levels were decreased in different degrees,especially in the DMD high-dose group(P<0.05).Compared with the blank group,the inflammatory factors tumor necrosis factor-α(Tumor necrosis factor-α,TNF-α),interleukin-6(Interleukin-6,IL-6),interleukin-2(Interleukin-2,IL-2),interleukin-1β(Interleukin-1β,IL-1β),inducible nitric oxide synthase(Inducible nitric oxide synthase,i NOS),gamma interferon(Interferon-γ,Compared with the model group,the levels of TNF-α,IL-6,IL-2,IL-1β,i NOS,IFN-γ in the pancreatic tissue of the rats in each administration group were significantly increased(P<0.05).The contents were decreased in different degrees,especially in the DMD high-dose group(P<0.05).It shows that the inflammationdominated pathological change mechanism of AP is closely related to the inflammatory cascade mediated by the HMGB1/TLR4/NF-κB pathway response.DMD can inhibit the expression of key molecular genes and proteins of this signaling pathway and reduce pancreatic tissue inflammation.reaction.4.Real-time quantitative polymerase chain reaction,western blotting and immunohistochemistry were used to detect the protein expression and phosphorylation level of key molecules of HMGB1/RAGE/AKT signaling pathway.Compared with the blank group,the receptor for advanced glycation endproducts(RAGE),Phosphatidylinositol 3 kinase(PI3K),and protein kinase B in the pancreatic tissue of the model group were significantly increased.(Protein Kinase B,AKT),B-cell lymphoma/leukemia-2(B-cell lymphoma/leukemia-2,Bcl-2)gene protein expression was significantly increased,cysteine protease 3(Caspase-3),The expression of Bcl-2Associated X Protein(Bax)gene protein was significantly decreased(P<0.05).The expression of gene and protein decreased in different degrees,and the expression of Caspase-3 and Bax gene protein increased in different degrees,especially in the DMD high-dose group(P<0.05).It shows that the apoptosis of pancreatic acinar cells in the rat AP model is inhibited,which may be related to the severe inflammatory necrosis after the onset of AP,and DMD can significantly inhibit the activation of PI3K/AKT pathway mediated by HMGB1/RAGE,thereby promoting acinar cells.Apoptosis process,reduce inflammatory necrosis,and play a protective role of pancreas.In this study,by rebuilding the rat AP model and carrying out related experiments,it is proved that DMD can intervene in the rat AP model through the two signaling pathways of "inflammation" and "apoptosis".The mechanism of action and therapeutic targets in the clinical treatment of AP enriches the theoretical basis for the clinical application of this prescription,and provides basic support for further exploration of this prescription and related TCM theories.