Study On Lianpo Yin Modified Decoction For Treatment Of Clarithromycin Resistant Helicobacter Pylori Associated Gastritis

Objectives1 To study the microbiological characteristics of Helicobacter pylori,clarify the relationship between Helicobacter pylori infection and dampness-heat syndrome of spleen and stomach in traditional Chinese medicine,and elucidate the rationality of treating Helicobacter pylori associated gastritis with Lianpo Yin（LPY）.2 To study the inhibiting effect of LPY modified decoction on clarithromycin-resistant Helicobacter pylori in vitro in comparison with six common antibiotics.3 To study the effects of LPY modified decoction on the body weight,small intestine length,concentrations of serum TNF-a and IL-6,and Warthin-Starry silver staining of gastric mucosa in mice with actinomycin-resistant Helicobacter pylori gastritis,and to compare it with the bismuth-containing quadruple therapies,and to explore the improvement mechanism of LPY modified decoction on the above indexes.4 To study the effect of LPY modified decoction on the metabolism of bile acid in mice with clarithromycin-resistant Helicobacter pylori associated gastritis,and to compare it with bismuth-containing quadruple therapies,and to explore the improvement mechanism of LPY modified decoction on the metabolism of bile acids5 To study the effect of LPY modified decoction on the intestinal flora in caecal contents of mice with clarithromycin-resistant Helicobacter pylori associated gastritis,and to compare it with bismuth-containing quadruple therapies,and to explore the improvement mechanism of LPY modified decoction on intestinal flora.Methods1 By summarizing and analyzing research results of Helicobacter pylori based on the traditional Chinese and western medicine,the characteristics of Helicobacter pylori in epidemiology,morphology,thallus dynamics,biochemistry,in vivo distribution,pathogenic mechanism and pathogenic mechanism theory were studied in detail.Therefore,the close relationship between Helicobacter pylori infection and dampness-heat syndrome of spleen and stomach in traditional Chinese medicine and the rationality of treating Helicobacter pylori associated gastritis with LPY were elucidated from two aspects of pathogenic microorganism’s own characteristics and immune response to host.2 Experimental research:Four gastric mucosa samples were collected from patients admitted to the Digestive Endoscopy Center of Optics Valley Hospital of Hubei Provincial Hospital of Traditional Chinese Medicine from November 2019 to September2020.The subjects were tested positive by ¹³C-urea breath test or ¹⁴C-urea breath test.Gastroscopic examination was consistent with chronic gastritis.The patients met the diagnostic criteria of dampness-heat syndrome of spleen and stomach in traditional Chinese medicine.One piece of gastric mucosa was collected for rapid urease test,and another piece of gastric mucosa was taken for drug resistance gene detection of Helicobacter pylori from positive individuals,and the remaining two pieces were cultured for Helicobacter pylori in vitro.After being identified as Helicobacter pylori,the concentration of Helicobacter pylori was adjusted to 0.5*10⁹to prepare the drug sensitive test blood plate.The drug sensitivity test was carried out with six kinds of antibiotic sheets and LPY modified decoction sheets by the diffusion method of paper sheets.The experiment was repeated for three times,and the average diameter of antibacterial circle of LPY modified decoction sheets was recorded and compared with the six kinds of antibiotics.Ninety SPF 6-week-old BALB/c mice,all male,were randomly divided into five groups:normal saline group（SL group）,pathological model group（BL group）,LPY modified decoction（ZY group）,bismuth-containing quadruple therapies group（XY group）,and combined treatment group（ZX group）.After adaptive feeding for six days,except normal saline group（SL group）,mice in each group were given 5×10⁹CFU/m L clarithromycin resistant bacterial solution 0.2 m L twice every other day for 19 days.Weight was recorded every two days.Two mice were randomly selected from each group for verification after the last gavage and routine feeding for 3 days.Positive urease test of gastric tissue and Warthin-Starry silver staining of gastric mucosa for Helicobacter pylori colonization were the signs of successful modeling.After successful modeling,each group was interfered for14 days.Normal saline group（SL group）and pathological model group（BL group）were given 0.3 m L of normal saline/day by gavage.LPY modified decoction group（ZY group）was given 0.5g/ml LPY modified decoction0.3ml/day by gavage;Bismuth-containing quadruple therapies group（XY group）was intragastrically administrated with normal saline solution of quadruple drugs（amoxicillin,levofloxacin,esomeprazole,bismuth potassium citrate）0.3ml/day;combined treatment group（ZX group）was intragastrically administrated with the solution of LPY modified decoction containing bismuth-containing quadruple therapies 0.3ml/day.Three days after the end of drug administration intervention,mice in each group were sacrificed and the length of small intestine was measured.The fixed gastric tissue was stained with Warthin-Starry silver.Blood was collected from orbital sinus to separate serum and the concentrations of IL-6and TNF-αwere determined by ELISA.The whole gallbladder was taken and frozen in liquid nitrogen at-80℃.The bile acids in the brain cysts were quantitated by liquid chromatography mass spectrometry.The cecal contents were collected and intestinal flora was detected by 16S r RNA sequencing technology.Results1 Helicobacter pylori is helical,unipolar flagellum structure,with high urea decomposition ability,these characteristics enable it to proliferate in gastric mucosa epithelium.Helicobacter pylori has strict requirements on colonization environment.The main pathogenic mechanism of Helicobacter pylori is immune damage,and its carcinogenic effect is related to gene errors in tissue repair.In terms of the microscopic dialectics of gastric mucosa and the overall symptoms of patients,the infection of Helicobacter pylori is highly consistent with the study on the nature of dampness-heat syndrome of spleen and stomach in traditional Chinese medicine.Traditional Chinese medicine has significant advantages in improving the microenvironment and regulating immunity.LPY has a significant effect on the treatment of Helicobacter pylori associated gastritis.On the basis of the original prescription,LPY modified decoction added drugs to inhibit bacteria and improve gastric mucosa,which can treat Helicobacter pylori associated gastritis from the characteristics of pathogenic microorganisms and host immune response.Our previous study showed that LPY modified decoction was in line with the potential pathway of reducing inflammation in terms of network pharmacology,could promote gastrointestinal motivity and enhance gastrin level,and had the mechanism of action in the treatment of Helicobacter pylori associated gastritis.2.1 Genetic tests showed that the selected Helicobacter pylori had mutations in clarithromycin and furazolidone resistance genes.The diameters of inhibition zones of clarithromycin,tetracycline,metronidazole,amoxicillin,furazolidone and leofloxacin were 9 mm,12 mm,4 mm,18 mm,20 mm and 23 mm respectively in the susceptibility test of paper diffusion method,and the selected Helicobacter pylori was resistant to clarithromycin,tetracycline and metronidazole.The diameter of the inhibition zone of LPY modified decoction with the concentration of 0.5g/m L was 17.78±1.64mm.2.2 In bismuth-containing quadruple therapies group（XY group）,spherical bacteria aggregation was observed in the gastric pits.A small amount of rod-shaped bacteria were scattered in the mucus of LPY modified decoction（ZY group）.2.3 Among the 43 bile acids,there was no statistical difference between the LPY modified decoction（ZY group）and the normal saline group（SL group）.The 13 bile acids with differences between groups were CUCA,αMCA,βMCA,λMCA,ωMCA,CA,ACA,7-ketod CA,TCDCA,TDCA,THDCA,TLCA,GUDCA.The contents of CUCA,αMCA,βMCA,λMCA andωMCA in bismuth-containing quadruple therapies group（XY group）were significantly higher than those in the pathological model group（BL group）.These results indicated that bismuth-containing quadruple therapies could significantly increase the contents of these five bile acids in clarithromycin-resistant HAG mice.The contents of CA,ACA and 7-ketod CA in bismuth-containing quadruple therapies group（XY group）were significantly higher than those in the pathological model group（BL group）,and the contents of LPY modified decoction group（ZY group）were significantly lower than those in bismuth-containing quadruple therapies group（XY group）.These results indicated that bismuth-containing quadruple therapies could significantly increase the contents of these three bile acids in clarithromycin-resistant HAG mice,which had different effects on these three bile acids compared with LPY modified decoction therapy.The content of TCDCA in bismuth-containing quadruple therapies group（XY group）was significantly lower than that in the pathological model group（BL group）.These results suggested that antibiotic quadruple therapy could significantly reduce the content of this bile acid in clarithromycin-resistant HAG mice.In terms of the content of TDCA,bismuth-containing quadruple therapies group（XY group）was significantly higher than the normal saline group（SL group）,and LPY modified decoction group（ZY group）was significantly lower than bismuth-containing quadruple therapies group（XY group）.There was no significant difference between LPY modified decoction group（ZY group）and the normal saline group（SL group）.The results indicated that bismuth-containing quadruple therapies would produce adverse disturbance to the bile acid,and LPY modified decoction therapy had more advantages in maintaining the physiological homeostasis of the bile acid.In terms of THDCA content,the LPY modified decoction（ZY group）and the four antibiotic groups（XY group）were significantly lower than that of normal saline group（SL group）,and the LPY modified decoction group（ZY group）was significantly higher than that of bismuth-containing quadruple therapies groups（XY group）.The results indicated that both LPY modified decoction therapy and bismuth-containing quadruple therapies could cause adverse disturbance to THDCA of bile acid,and the disturbance effect of LPY modified decoction group therapy was weaker,which was superior than bismuth-containing quadruple therapies.The contents of TLCA and GUDCA in combined treatment group（ZX group）and bismuth-containing quadruple therapies group（XY group）were significantly lower than those in normal saline group（SL group）,and LPY modified decoction group（ZY group）was significantly higher than that in bismuth-containing quadruple therapies（XY group）.Both bismuth-containing quadruple therapies and LPY modified decoction group combined with bismuth-containing quadruple therapies may cause adverse disturbance to bile acid TLCA and GUDCA.LPY modified decoction group combined with bismuth-containing quadruple therapies cause less disturbance to bile acids TLCA and GUDCA.Combination of LPY modified decoction helps to reverse the bad disturbance of bile acid caused by bismuth-containing quadruple therapies.2.4 In mice infected with clarithromycin-resistant Helicobacter pylori,the abundance of intestinal flora decreased significantly（P<0.05）,the strains and content in the normal saline group（SL group）were the most similar with LPY modified decoction group（ZY group）;The abundance of bacteria in the bismuth-containing quadruple therapies group（XY group）decreased significantly,and the dispersion of samples was the highest.Combined treatment group（ZX group）was between the two groups.A total of 2,043 OTUs were produced by the intestinal flora of the caecal contents of the five groups of mice,and 1,952 OTUs remained after the leveling treatment.Compared with the normal saline group（SL group）,the number of overlapped OTUs was 635 in the antibiotic quad group（XY group）,783 in the combined treatment group（ZX group）,and 787 in the LPY modified decoction group（ZY group）.The number of OTUs overlapping with normal saline group（SL group）in LPY modified decoction group（ZY group）and bismuth-containing quadruple therapies group（XY group）was significantly higher than that in bismuth-containing quadruple therapies group（XY group）.The results indicated that the cecal intestinal flora of mice with bismuth-containing quadruple therapies was significantly different from that of mice with normal saline intragastric administration,and the combination of traditional Chinese medicine on the basis of antibiotic quadruple therapy could restore the intestinal flora to a certain extent.From the perspective of intestinal flora,the treatment of clarithromycin resistance HAG by LPY modified decoction is most beneficial to maintaining intestinal flora homeostasis.Conclusions1 Theoretical research:LPY in the treatment of Helicobacter pylori associated gastritis is consistent with the principle of prescription and syndrome corresponding,and is reasonable.2 In vitro experiments,LPY modified decoction had inhibitory effect on clarithromycin resistant Helicobacter pylori,which was similar to the antibacterial effect of amoxicillin.3 In mice,LPY modified decoction has inhibitory effect on clarithromycin resistant Helicobacter pylori,and can reduce the resistance of clarithromycin resistant Helicobacter pylori to antibiotic quadruple therapy by reducing the spherical change.4 Compared with bismuth-containing quadruple therapies,LPY modified decoction group has less disturbance to gallbladder bile acid,and has significant advantages in maintaining the physiological homeostasis of bile acid.5 Compared with antibiotic quadruple therapy,LPY modified decoction has less disturbance to the abundance of intestinal flora,and has a significant advantage in maintaining the homeostasis of intestinal species diversity.6 LPY modified decoction has a significant effect on clarithromycin resi stant Helicobacter pylori associated gastritis.Compared with bismuth-con taining quadruple therapies,there is no spherical change of drug resistan ce phenomenon.It also has significant advantages in maintaining bile aci d metabolism homeostasis and maintaining intestinal species diversity.