| Objiect:1 Summarize the related theoretical concepts of warm disease epidemic,sorting out the classification,diagnosis and treatment of epidemic diseases,exploring the connotation of TCM theory of COVID-19.Through GNT to clear the TCM treatment of COVID-19 based on the theory of dampvirus epidemic,and uncover new approaches for treating infections diseases.2 We conducted retrospectively clinical cases of GNT,and evaluate its efficacy of COVID-19.3 To explore the therapeutic targets and pathways of GNT by using modern information and experimental technology,for which we investigate its mechanism in inhibiting the formation of neutrophil extracellular traps(NETs)through MAPK/JNK pathway and reducing the inflammatory response of acute lung injury.Methods:1 Theoretical research(1)Though reading ancient books and literature to sort out the formation and development of the theory of warm disease,review and study the development,modern connotation and classification of warm disease,clarify the concept and classification of epidemic diseases,and analyze the syndrome and treatment of dampness virus epidemic.(2)Exploring the pathogenesis,syndrome and treatment of COVID-19 based on the theory of warm disease,and explain the modern mechanism of TCM by our previous research.2 Clinical research(1)231 COVID-19 patients were selected for inclusion,who admitted to Wuhan Third Hospital and Hubei Provincial Hospital of Traditional Chinese Medicine from January 27,2020 to March 18,2020 were enrolled.According to the treatment plan,the patients were divided into intervention group 1(Guanning decoction group,n =83),intervention group 2(Qingfei Paidu decoction group,n = 101)and western medicine group(n = 47).We analyzed their gender,age,general conditions,and whether they had underlying diseases and Sars-cov-2 PCR positive.Summary the initial symptoms and lung CT of the disease to prove the etiology and pathogenesis.(2)The average length of hospital stay,the rate of transfer to ICU during the observation period,the incidence of complications and mechanical ventilation,the cure rate,the improvement rate,the mortality rate,the negative conversion time of nucleic acid,the CT outcome,the improvement of symptoms and the changes of laboratory indicators of the three groups were analyzed,and the differences in clinical outcomes between the integrated traditional Chinese and western medicine and western medicine treatment were analyzed.3 Experimental study(1)Liquid chromatography tandem mass spectrometry(LC-MS/MS)technology was used to identify some components of GNT.The enrichment of the targets and pathways of GNT reveal its mechanisms and potential therapeutic targets.(2)Research on anti-inflammatory mechanism.LPS-induced acute lung injury mouse models was established.Fifty-six mice were randomly divided into blank group(Control),Model group(Model),methylprednisolone group(MPN),Guanning decoction equal dose group(GNT),Guanning decoction high dose group(GNTH),Guanning decoction equal dose + methylprednisolone group(GNT+MPN),Guanning decoction high dose + methylprednisolone group(GNTH+MPN).There were 8 rats in each group,4 males and 4 females.After 3 days of drug intervention,mice were smoke LPS to establish the model.The mice were given gavage once after an hour modeling.Samples were taken after a 7-hour fasting and water deprivation period following the last gavage.HE staining was used to observe the local changes and inflammatory infiltration of lung tissue.The levels of IL-1β,IL-6,and TNF-α in bronchoalveolar lavage fluid(BALF)and serum were detected by ELISA,and the levels of IL-1β,IL-6,and TNF-α in lung tissue were detected by qPCR.We therefore started to explore the potential beneficial effects of anti-inflammatory of GNT in models of ALI mice.(3)There we use LPS-induced ALI mouse models too.Forty-eight mice were randomly divided into blank group(Control),Model group(Model),methylprednisolone group(MPN),Guanning decoction equal dose group(GNT),GSK484 group(PAD4 inhibitor group),SP600125group(JNK pathway inhibitor group),with 8 mice in each group,4males and 4 females.The Control and the Model group were given normal saline at 0.01ml/g by gavage,the GNT group was 2-fold diluted standard solution at 0.01ml/g by gavage,the MPN group was given drug at 0.01ml/g by gavage,the SP600125 group was given0.01ml/g by tail vein injection,and the GSK484 group was given0.01ml/g by intraperitoneal injection.The drug was given once daily for 3 consecutive days.On day 3,modeling was started 1 hour after administration.One hour after the end of modeling,each group received another dose of medication.After the last administration,all mice were fasted and water deprived for 7 hours,and the mice were sacrificed for sampling.The formation of NETs(inflammatory cell infiltration)in the pathological sections of the lungs was observed under a microscope.The levels of NE and MPO in bronchoalveolar lavage fluid(BALF),serum and lung tissue were detected by ELISA.To explore the anti-inflammatory protective effect of GNT in ALI mice.Immunofluorescence staining was used to measure MPO,and the ratio score of MPO cells was calculated to measure the degree of NETosis in tissues.The levels of PAD4,JNK and p-JNK were detected by Western blot to observe the effect of GNT on the pathways.Results:1 Theoretical results(1)Warm epidemic is not a specific disease.Any warm disease with rapid onset,rapid transmission,dangerous condition,strong infectivity and widespread epidemic can be called warm epidemic.Warm epidemic is caused by the feeling of epidemic pestilence,and it is not different from warm disease in nature.When the epidemic of warm disease occurs in a large range,it can be called warm epidemic.Therefore,compared with the general warm disease,there is no obvious seasonality.(2)No matter what TCM nomenclature of COVID-19 is,most of the nomenclature reflects that the characteristics of the disease are related to "dampness".Our research group believes that the main disease is damp-heat toxin.In the early stage,"dampness and toxics" occurred,and then "blood-stasis and qi-block" appeared.The treatment is mainly to eliminate dampness and heat and Du-toxin,and unblock qi.It is necessary to make early diagnosis,focus on toxin,and prevent transmission.(3)COVID-19 can cause ALI,which NETs may be involved in the excessive inflammatory response of ALI.Traditional Chinese medicine treatment can improve the prognosis of the disease.2 Clinical results(1)GNT can alleviate the clinical symptoms and improve the laboratory indexes of patients,increase the HGB,LYM,ALB,and decrease the NLR,AST,CK,CRP,LDH,PLR compared with before.(2)GNT group had lower rate of ICU transfer,complications and mechanical ventilation.3 Experimental results(1)GNT results levamisole,glycine,betaine,citrate,gluconic acid,proline,Tengaligtonone,glycyrrhizin H2,L-choline,flavonol,amygdalin,DL-3-phenyllactic acid,asphotomoside BII,polamine,isoleucine,phenylpyruvate,seranoglycoside C,furoside,new sapogenin 3-[glucosyl-(1-1->2)-glucosyl-(1->4)-galactoside],1-(4-hydroxy-2-methoxyphenyl)-ethyl ketone,Baicalein flavone Ⅱ,valine,3-hydroxycoumarin and other active ingredients,these ingredients can participate in anti-inflammatory,metabolic and other biological functions.Network pharmacology was used to find the components and targets of GNT.GO function analysis and KEGG pathway analysis showed that GNT had a high role in MAPK pathway and proteins.It possible could reduce inflammatory indicators and have anti-inflammatory effect,which was confirmed in the clinical study.There were 34 targets of GNT,such as JUN,CASP3,MAPK3,MAPK1,MAPK14,TNF,AKT1 and STAT3 in this pathway.MAPK is related to gene expression regulation and cytoplasmic functional activity.The top10 genes of GNT were closely related to MAPK pathway.In particular,MAPK3,AKT1 and JUN are closely related to JNK in the MAPK pathway,so we speculate that the action pathway of GNT may be MAPK/JNK.(2)The results on anti-inflammatory mechanism: we used a microscope to observe the pathological sections of the lung after HE staining at 200 magnification.In the Control group,the overall structure of lung tissue was basically normal,the alveolar structure was clear,there was no alveolar atrophy,expansion and other degeneration,no obvious exfoliation and necrosis of alveolar epithelial cells,no obvious congestion and expansion of pulmonary interstitium,and no obvious inflammatory cell infiltration in alveolar cavity.In the Model group,the overall structure of lung tissue was abnormal,with a large number of alveolar atrophy and collapse,obvious thickening of alveolar wall,and large area of parenchymal change.There was no obvious congestion and expansion of blood vessels in the pulmonary interstitium,and obvious inflammatory cell infiltration in the alveolar cavity.In GNT group,the overall structure of lung tissue was abnormal,with a large number of alveolar atrophy and collapse,obvious thickening and local parenchymal alveolar wall,no obvious blood vessel congestion and expansion in pulmonary interstitium,and a small amount of inflammatory cell infiltration in alveolar cavity.In GNTH group,the overall structure of lung tissue was abnormal,and there was no alveolar fusion,expansion,atrophy and other degeneration.Some alveolar epithelial cells were shed,a small amount of alveolar wall was slightly thickened,and a small amount of inflammatory cell infiltration was observed in alveolar cavity.The overall structure of lung tissue in MPN group was abnormal,and there was no alveolar fusion,expansion,atrophy and other degeneration,but a small amount of alveolar wall slight thickening and obvious inflammatory cell infiltration in alveolar cavity were observed.In GNT+MPN group,the overall structure of lung tissue was slightly abnormal,there was no alveolar fusion,expansion,atrophy and other degeneration,no obvious thickening of alveolar wall,and obvious inflammatory cell infiltration in alveolar cavity.In GNTH+MPN group,the overall structure of lung tissue was slightly abnormal,there was no alveolar fusion,expansion,atrophy and other degeneration,no obvious thickening of alveolar wall,and obvious inflammatory cell infiltration in alveolar cavity.The levels of IL-1β,IL-6,and TNF-α in bronchoalveolar lavage fluid(BALF)and serum were detected by ELISA.Compared with the Model group,the levels of IL-1β,IL-6,and TNF-α in the bronchoalveolar lavage fluid and the levels of IL-1β,IL-6,and TNF-α in the serum were decreased,and the differences were statistically significant(P<0.001).After treatment with MPN and GNT,the inflammatory indexes of the mice were all decreased,with statistical significance(P<0.001).GNT,GNTH,GNT+MPN and GNTH+MPN decreased more than MPN,and GNTH decreased the most,followed by GNT+MPN and GNTH+MPN groups.The difference between the two groups was statistically significant(P<0.001).The levels of IL-1β,IL-6,and TNF-α in lung tissues were detected by qPCR.Compared with the Model group,the levels of IL-1β,IL-6 and TNF-α in lung tissue were decreased in each group,and the differences were statistically significant(P<0.01).After the intervention of MPN and GNT,the inflammatory indicators in lung tissue of mice were decreased,with statistically significant differences(P<0.001),and the degree of decrease in MPN,GNT+MPN and GNTH+MPN was greater than that in GNT and GNTH.GNT+MPN and GNTH+MPN decreased most significantly,followed by MPN group.The difference between the two groups was statistically significant(P<0.001).There was no significant difference in inflammatory markers in GNT and GNTH lung tissues(P>0.05).(3)Study on anti-inflammatory and protective pathways.HE pathological sections were observed under a microscope of 200 times.In the Control group,the overall structure of lung tissue was basically normal,the alveolar structure was clear,there was no alveolar atrophy,expansion and other degeneration,no obvious exfoliation and necrosis of alveolar epithelial cells,no obvious congestion and expansion of pulmonary interstitium,and no obvious inflammatory cell infiltration in alveolar cavity.In the GNT group,the overall structure of lung tissue was abnormal,no alveolar fusion,expansion,atrophy and other degeneration were observed,some alveolar walls were significantly thickened and slightly parenchymal,no vascular congestion and expansion were observed in the pulmonary interstitium,and a small amount of inflammatory cell infiltration was observed in the alveolar cavity.However,the MPN group showed obvious inflammatory cell infiltration.In the PAD4 inhibitor group,the overall structure of lung tissue was slightly abnormal,there was no alveolar fusion,expansion,atrophy and other degeneration,no obvious thickening of the alveolar wall,no obvious congestion and expansion of blood vessels in the pulmonary interstitia,and obvious inflammatory cell infiltration in the alveolar cavity.In SP600125 group,the overall structure of lung tissue was basically normal,the alveolar structure was clear,there was no alveolar atrophy,expansion and other degeneration,no obvious exfoliation and necrosis of alveolar epithelial cells,no obvious congestion and expansion of pulmonary interstitium,and a small amount of inflammatory cell infiltration in alveolar cavity.ELISA was used to detect the levels of NE and MPO in BAFL,serum and lung tissue.Campare with those in the Model group,the levels of NE and MPO were lower in others.The reduction level in MPN group was greater than that in GNT group,but less than that in GSK484 and SP600125 groups,and the difference was statistically significant(P<0.01).The ratio score of MPO cells measured by immunofluorescence staining showed that the percentage of MPO positive cells in each group was lower than that in Model group.The levels of PAD4,JNK and p-JNK were measured by WB immunoblotting.After intervention,compared with the Model group,the relative values of PAD4/β-actin,JNK/β-actin and p-JNK/β-actin in the other groups decreased,except for the MPN group,the other groups decreased significantly(P<0.01).GNT can reduce the levels of NE and MPO in BALF,serum and lung tissue of ALI mice,and also reduce the protein level of peptidylarginine deiminase(PAD4),and inhibit the phosphorylation of JNK.Conclusions:1 According to clinical manifestations of COVID-19 patients,the disease is characterized by damp-heat toxicosis,which can also be called damp-heat epidemic toxin.In the early stage,the disease is characterized by "dampness and toxics ",and then the appearance of " blood-stasis and qi-block ".COVID-19 is a dampness virus epidemic,and the treatment is mainly to eliminate dampness and heat epidemic virus and to clear qi.It is necessary to make early diagnosis,focus on exorcizing pathogens,and prevent transmission.2 Traditional Chinese medicine can improve the clinical symptoms of patients.GNT can improve and increase the levels of HGB,LYM and ALB in patients with COVID-19,reduce the levels of CK,CRP,LDH and PLR,and play a protective role for patients.The combination of Chinese and western medicine has advantages in the treatment of COVID-19.3 GNT can reduce the levels of IL-1β,IL-6,TNF-α,NE and MPO in BALF,serum and lung tissue of ALI mice.It can reduce the phosphorylation of JNK,inhibit NETosis and reduce inflammatory response by reducing the level of PAD4 protein.The anti-inflammatory mechanism is achieved through the MAPK/JNK pathway. |
PDF Full Text Request