| Background:Gastric ulcer(GU)is a common digestive tract and frequently-occurring disease.The symptoms of GU can be relieved after drug treatment,but its curability is low and easy to repeat.As a kind of unique traditional Chinese medicine,charcoal medicine has a long history and is widely used in the treatment of gastric diseases,especially gastric ulcers,with remarkable curative effect.Nelumbinis Rhizomatis Nodus carbonisata(NRNC)is the processed product of Nelumbinis Rhizomatis Nodus(NRN),and was first recorded in Song dynasty "Wan Ji shang".The effect of NRNC is "convergence and hemostasis,removing blood stasis,and treat for various bleeding disorders",such as "hemoptysis","hemoptysis" and other digestive tract lesions.However,there is no report on the material basis and pharmacodynamic mechanism of NRNC in the treatment of peptic ulcer with bleeding characteristics,which largely limited its wide clinical application.In the previous research,our team,under the guidance of the theory of traditional Chinese medicine,used the pharmacodynamic model and the technical methods in the field of nanoscience to explore the material basis of charcoal medicine from the perspective of nanopharmacology,which was a new kind of physicochemical properties similar to nanoparticles substances,and named them nano-components(NCs).This provides a research idea that can be used for reference in this paper.Therefore,this study intends to evaluate whether the pharmacodynamic material basis of NRNC in the treatment of gastric ulcer is NCs,and to explore its mechanism of action.Objective:1.NRNC-NCs were extracted and isolated from commercially available NRNC,which were preliminarily confirmed as the material basis of anti-gastric ulcer.2.The optimal fabrication process parameters of NRNC-NCs were optimized and systematically characterized in laboratory conditions.3.The ethanol-induced acute gastric ulcer in rat and stress gastric ulcer models were used to explore the efficacy of NRNC-NCs against gastric ulcer and its mechanism of action.4.The safety of NRNC-NCs was evaluated,which can provide a scientific basis for their clinical application dosage and safe.Method:1.The classical ethanol-induced rat acute gastric ulcer model was used to compare the NRN,market NRNC(MRNRC),MRNRC dialysis bag outside solution(MRNRC-O)and MRNRC dialysis bag inside solution(MRNRC-I)on acute gastric ulcer in rats.The ulcer index was calculated,and the histopathological changes were evaluated,which were determined the effective part in NRNC to improve gastric ulcer.Furthermore,the structural and optical characteristics of the effective part in NRNC were characterized with the help of nano-characterization techniques and spectroscopic techniques,such as Transmission Electron Microscopy(TEM),UV-vis Absorption Spectroscopy(UV-vis)and Fluorescence Spectrum(FL)and Fourier transform infrared spectroscopy(Fourier transform Infrared.FTIR).2.The processing method of NRNC was optimized,and NRNC-NCs were prepared at different temperatures(300℃,350℃,400℃,450 ℃),and their morphological structures,surface groups and fluorescence characteristics were characterized and analyzed.The optimal preparation parameters of NRNC-NCs were screened with ethanol-induced acute rat gastric ulcer model,which were screened and further characterized.3.The protective effect of NRNC-NCs prepared under optimal conditions on ethanolinduced human gastric mucosal epithelial cells(GES-1)was evaluate in vitro.Furthermore,the protective effect and mechanism of action of NRNC-NCs on gastric mucosal injury in rats with ethanol-induced gastric ulcer was explored in vivo.The gastric ulcer index and gastric histopathology were calculated and examined.The SOD,CAT,GSH-Px,GSH and MDA content and TNF-α,IL-6 and EGF levels were detected in gastric tissue.Moreover,the expressions of NF-κB,COX-2,VEGF and AKT were analyzed by immunohistochemistry.The protein expression of NF-κB was further verified with immunofluorescence analysis.Finally,the related expression of NF-κB p65 in the cytoplasm and nucleus and PI3K,AKT and Cleaved-caspase 3 were analyzed by Western blotting(WB)in gastric tissue.In addition,this study combined non-target-metabolomic methods to search for potential metabolic markers in serum samples of rats in ethanol-induced gastric ulcer,which explore the potential mechanism of NRNC-NCs in improving gastric ulcer in rats.4.To evaluate the effects of NRNC-NCS on gastric ulcer index and pathological changes in stress ulcer rats.At the same time,the SOD.MDA.TNF-α,IL-6 and EGF levels in gastric tissue of rats were detected,and the levels of DA and 5-HT in rat brain and serum were further determined,which preliminarily revealed the efficacy and mechanism of NRNCNCS on stress gastric ulcer rats.5.The safety of NRNC-NCs was preliminarily evaluated by the acute toxicity and subacute toxicity experiments.The general signs,mortality,body weight and organ index were monitored and calculated in mice.Hematological,biochemical and pathological changes were also detected to investigate the toxicity of NRNC-NCs.Result:1.NRN,MNRRC,MNRRC-O and MNRRC-I have anti-ethanol-induced gastric ulcer effect in rats.Among them,the antigastric ulcer effect of MNRNC is stronger than that of NRN.Moreover,the result showed that the anti-gastric ulcer effect of MNRNC-I is stronger than that of MNRNC-O.The order of activity of the above drugs against ethanol-induced gastric ulcer in rats is MNRNC-I>MNRNC>NRN>MNRNC-O,which indicated that the internal part of the dialysis bag was the main effective part of MNRNC.The results of characterization and analysis of the effective part solution showed that the structure of the effective part was spherical,the average particle size was 2.40±0.67 nm,the lattice spacing was 0.22 nm,and the surface contained functional groups such as hydroxyl,carbonyl and carboxyl groups.Based on the previous research foundation of the team,combined with the above characterization results,which was confirmed that it is a nano-component,and this paper named it as a lotus root carbon nano-component(NRNC-NCs).2.The characterization results show that there are some differences in the particle size of NRNC-NCs prepared at different temperatures.The UV-vis spectrum shows that NRNC-NCs prepared at 300℃ and 350℃ have UV absorption at 230-270 nm.FL detection showed that the maximum excitation wavelength and emission wavelength of NRNC-NCs prepared at different temperatures were different.In addition,the FITR results indicated that the surface chemical groups of NRNC-NCs prepared at different temperatures were mainly hydroxyl,carbonyl,and amino groups,but there were differences in peak absorption intensity.The efficacy evaluation confirmed that the NRNC-NCs prepared at different temperatures had the anti-ethanolic gastric ulcer effect in rats,and the NRNC-NCs prepared at 350℃ had the best effect.The NRNC-NCs prepared at 350℃ were further characterized in detail,and the results showed that they had an amorphous carbon structure,and the surface elements were mainly composed of C,O,and N with abundant surface groups,excellent dispersion and stability.The quantum yield of NRNC-NCs was 1.28%.3.The results of in vitro experiments show that NRNC-NCs have great biosafety,and they have repair and protection effects on GES-1 cells damaged by ethanol.In vivo experiments demonstrated that pretreatment of NRNC-NCs could reduce gastric ulcer index.increase ulcer inhibition rate.HE staining showed that pre-administration of NRNC-NCs could alleviate gastric hemorrhage,edema,and inflammatory infiltration in rats,which indicated that it had protective effect on gastric mucosa injury induced by ethanol in rats with gastric ulcer.The mechanism of action study indicated that pretreatment of NRNC-NCs could increase the levels of antioxidant stress factors(SOD.CAT,GSH-Px and GSH)in gastric tissue,decrease MDA and pro-inflammatory factors(TNF-α,IL-6),increase the growth factor EGF,up-regulate the protein expressions of VEGF,AKT and PI3K and down-regulate the protein expressions of Cleaved-caspase3,NF-κB and COX-2.This suggests that the mechanism of action of NRNC-NCs against ethanol-induced gastric ulcer in rats may be related to the improvement of gastric mucosal injury,antioxidant and anti-inflammatory systems.In addition,non-target-metabolomic results showed that NRNC-NCs may attenuate ethanol-induced acute gastric mucosal injury in rats by regulating lipid metabolism in rats.4.Pretreatment with NRNC-NCs can significantly reduce the gastric ulcer index,increase the ulcer inhibition rate in rats with stress gastric ulcer.The results also show that NRNC-NCs can significantly increase the levels of SOD and EGF in gastric tissue,reduce MDA,TNF-α,the level of IL-6.In addition,it can also reduce the content of DA and 5-HT in rat brain tissue and serum,thereby improving stress gastric ulcer in rats.5.The results of the acute toxicity experiment showed that the mice at each dose of NRNC-NCs did not die.Compared with the blank group,there was no difference in body weight,organ index,hematological index and biochemical index.When the maximum tolerated dose of NRNC-NCs was 200 mg/kg orally,the mice did not have any toxic reaction,and there was no difference between the detection indicators and the blank group.In the subacute toxicity experiment,there was no difference in body weight,organ index,hematological and biochemical indexes of mice in each dose group of NRNC-NCs during the administration period and the withdrawal recovery period.In addition,the results of HE staining during the administration period showed that the main organs were all within the normal range.During the recovery period of drug withdrawal,compared with male mice in blank group,the content of HGB and LDLC in male mice administered with NRNC-NCs(24 mg/kg)increased(P<0.05).Conclusion:This study is the first to explore that NRNC has significant anti-gastric ulcer activity in rats,and confirmed that its effective material basis is NRNC-NCs.NRNC-NCs have significant protective effects on the gastric mucosa of rats with ethanol-induced acute gastric ulcer and stress gastric ulcer.In ethanol-induced acute gastric ulcer,NRNC-NCs can accelerate gastric ulcer healing,increase antioxidant stress factors in rat gastric tissue,reduce inflammation,regulate NF-κB,PI3K/AKT signaling pathways and lipid metabolism,which alleviate gastric mucosal injury in rats.Meanwhile,NRNC-NCs can improve stress ulcer in rats by anti-oxidation and reducing inflammation of stomach tissue,and regulating the secretion of DA and 5-HT in brain tissue and serum.Moreover,NRNCNCs showed high safety in both acute toxicity and subacute toxicity tests,which provided a reference for clinical safe drug dosage.This study lays an experimental foundation for improving the clinical efficacy of NRNC in the treatment of gastric ulcer and for the development of corresponding new drugs. 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