| Background:Osteoporosis has a high incidence in patients with chronic pancreatitis.Osteoporosis is characterized by insidious onset and is usually only detected during physical examination or when brittle fractures occur.Hip and spine fractures,in particular,can lead to disability and inability to take care of themselves,placing a heavy burden on families and society.In addition,the firstline drugs used to treat osteoporosis have a lot of complications,and the overall treatment effect is not ideal.At present,the research on osteoporosis in chronic pancreatitis is mostly in clinical trials,and few people study the pathogenesis of osteoporosis in chronic pancreatitis from the perspective of animal experiments.According to the characteristics of chronic pancreatitis,it can be classified into the categories of "abdominal pain","epigastric pain" and "Pi Xin pain" in traditional Chinese medicine.The disease is "evil in the gallbladder,adverse to the stomach,damaging the liver and spleen" and should be treated mainly from the liver and spleen and from the gallbladder and stomach.At the same time,Qingyi Granule is an in-hospital preparation of The First Affiliated Hospital of Dalian Medical University,with the effects of soothing liver,promoting the function of gallbladder,relieving heat,relieving urgency and pain,which is a specific drug for the treatment of actute pancreatitis in our hospital.However,the curative effect and molecular mechanism of Qingyi Granule in treating osteoporosis in chronic pancreatitis are still unclear.Aim:1.To explore the molecular mechanisms of osteoporosis induced by chronic pancreatitis.2.Use ultra-high-performance liquid chromatography high-resolution mass spectrometry system to study the metabolic disorder caused by chronic pancreatitis and its association with the pathogenesis of osteoporosis.3.Exploration of the curative effect mechanism and metabolic mechanism of Qingyi Granule in intervening osteoporosis caused by chronic pancreatitis.Materials and methods:1.The mouse model of chronic pancreatitis was established by intraperitoneal injection of caerulein.Sixty male C57BL/6 mice were randomly divided into a control group(Con(N=30)and a chronic pancreatitis osteoporosis group(CP(N=30)).Chronic pancreatitis and osteoporosis model were established by intraperitoneal injection of 50 μg/kg caerulein six times a day at an interval of one hour and three days a week.Pancreas tissue,serum,femur,and feces were collected at 4,6,and 8 weeks,respectively.The pancreatic tissue was divided into two parts,and a part of the pancreatic tissue was fixed in paraformaldehyde for HE and Masson staining.The other part of the pancreatic tissues was quickly frozen in liquid nitrogen and placed in a-80 refrigerator for quantitative realtime reverse transcriptase PCR and Western Blot analysis as well as metabolomics and lipidomics.ELISA was used to detect the expression of transforming growth factor β(TGF-β),bone alkaline phosphatase(BALP),tartrate-resistant acid phosphatase(TRACP-5b)and fecal elastase-1.The mouse femoral tissue was divided into two parts.A part of the femoral tissue was fixed with paraformaldehyde and subjected to Micro-CT scan,HE and TRAP staining.The other part of the femur was quickly frozen in liquid nitrogen and used for Western Blot test.2.Ultra-high-performance liquid chromatography high-resolution mass spectrometry system is used for metabolomics and lipidomics detection.Week 8 mice Con(N=10)and CP(N=10)were selected for metabonomics and lipidomics of pancreatic tissue,serum and femur,respectively.The metabolic molecules were identified and resolved by the accurate molecular mass information of excimer ion peaks and fragment ions provided by the ultra-performance liquid chromatography-tandem high-resolution mass spectrometry system combined with the reference library comparison.3.Qingyi Granule intervention on chronic pancreatitis osteoporosis mouse model.Thirty male C57/BL6 mice were randomly divided into the control group Con(N=10),the chronic pancreatitis osteoporosis group CP(N=10)and the Qingyi Granule treatment group QYKL(N=10).The chronic pancreatitis model was also established by intraperitoneal injection of caerulein.The osteoporosis model of chronic pancreatitis was induced after continuous injection for 8 weeks.The rats in the Qingyi Granule group were treated with Qingyi Granule(1.3 g/kg)once in the morning and once in the evening after the second week of modeling.The rats in the control group and the osteoporosis group due to chronic pancreatitis were given the same volume of water by gavage.The pancreatic tissue,serum,femur and feces were also taken to explore the mechanism of Qingyi Granule in intervening with osteoporosis in chronic pancreatitis.In addition,pancreatic tissue samples were collected for metabolomics and lipidomics analysis using an ultra-performance liquid chromatography-tandem high-resolution mass spectrometry system to elucidate the metabolic mechanism of Qingyi Granules in the treatment of osteoporosis in chronic pancreatitis.Results:1.Intraperitoneal injection of caerulein establishes a mouse model of chronic pancreatitis and leads to osteoporosis.Four weeks after intraperitoneal injection of caerulein,HE and Masson staining of the pancreas showed pathological damage and extensive pancreatic fibrosis.Fecal elastase-1 was significantly reduced,and serum TGF-β level was increased.The expression levels of a-smooth muscle actin(a-SMA),Collagen and fibronectin 1 were also increased.In addition,Micro-CT showed significant decreases in trabecular bone thickness and junction density,as well as significant decreases in bone mineral density(BMD),trabecular bone thickness(Tb.Th)and bone surface area density(BS/TV)after 8 weeks of intraperitoneal injection of caerulein.Bone HE staining showed a decrease in trabecular bone thickness,and TRAP staining showed an increase in the number of osteoclasts.Compared with the control group,the expression levels of serum TGF-β,BALP and TRACP-5b were increased in the model group at week 8,the levels of TGF-β,nuclear factor κB(NF-κB)and receptor activator of nuclear factorκB ligand(RANKL)in the mouse femur were increased,and the levels of tumor necrosis factor receptor factor 3(TRAF3)and osteoprotegerin(OPG)were decreased.2.The unique metabolomics and lipidomics characteristics of osteoporosis mice with chronic pancreatitis.In pancreatic tissues,metabolites related to glycolysis and Krebs,including Glucose,Glucose 6-phosphate,phosphoglyceric acid,Phosphoenolpyruvic acid,pyruvic acid,Oxalacetic acid,cis-Aconitic acid,2-Oxoglutarate and succinic acid were up-regulated in the osteoporosis group with chronic pancreatitis,while fumaric acid and malic acid were down-regulated.Ceramide was down-regulated in the model group,while diacylglycerol,ethertype phosphatidylcholine and ether-type lysophosphatidylcholine,ether-type phosphatidylethanolamine,glycosphingolipid,sphingomyelin and triacylglycerol were up-regulated.In serum,glucose,lactic acid,citric acid,cis-aconitic acid,fumaric acid and malic acid were up-regulated in the osteoporosis group due to chronic pancreatitis.Long-chain acylcarnitine,ceramide,ether-type phosphatidylcholine,glycosphingolipid,sphingomyelin and triacylglycerol with high levels of unsaturation are down-regulated,while triglyceride and diacylglycerol with high degree of saturation are up-regulated.3.Qingyi Granules can alleviate the degree of pancreatic fibrosis and inflammation,and reduce osteoporosis.Compared with the osteoporosis group with chronic pancreatitis,Qingyi Granules could reduce the expression levels of serum TGF-β,BALP and TRACP-5b,and increase the content of Fecal elastase1.HE and Masson staining of pancreatic tissue showed that Qingyi Granule could significantly reduce the pathological damage and fibrosis degree of pancreatic tissue.Qingyi Granule could significantly down-regulate the content of a-SMA,Collagenl and Fibronectinl and their mRNA expression levels in pancreatic tissues.Micro-CT scan showed that the thickness and junction density of trabecular meshwork were significantly increased in the QYKL group,while the BMD,Tb.Th and BS/TV were significantly increased.Western Blot analysis of the femur showed that compared with the CP group,Qingyi Granules significantly down-regulated the expression levels of TGF-β,RANKL and NF-κB in the femur,and increased the expression levels of TRAF-3 and OPG.Metabolomics and lipidomics results of pancreatic tissues showed that Qingyi Granules could reverse the polar metabolite and lipids changes in the chronic pancreatitis osteoporosis group.Conclusions:The mouse model of chronic pancreatitis with osteoporosis could be established by intraperitoneal injection of caerulein for 8 weeks.It also verified that TGF-β/TRAF-3/NF-κB/RANKL pathway mediated osteoporosis in chronic pancreatitis,and Qingyi Granule could reduce the pathological damage and fibrosis degree of pancreatic tissue,and it might regulate TGF-β/TRAF-3/RANKL pathway to treat osteoporosis in chronic pancreatitis.Non-targeted metabolomics and lipidomics explored the metabolic disorders caused by chronic pancreatitis and its association with the pathogenesis of osteoporosis,and the metabolic mechanism of Qingyi Granule in intervening with osteoporosis caused by chronic pancreatitis. |
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