Clinical And Experimental Study On Treating Agkistrodon Halys Bite With 717 Jiedu Mixture

1 Objective1.1 To observe the clinical effect of 717 Jiedu Mixture on patients with Agkistrodon halys bite,blood biochemical values such as Myocardial zymogram(CK,CK-MB),liver function indicators(AST,ALT),renal function indicators(BUN,CREA),blood routine(WBC,CRP),blood coagulation function(APTT,PT),inflammatory mediators(TNF-α,IL-6,5-HT,Histamine)and other values to be tested,To explore the mechanisms of 717 Jiedu mixture in the treatment of Agkistrodon halys bite..1.2 Proteomics observation of the differential protein expression in the wound tissue of agkistrodon-injured rats: The gastrocnemius muscle wound tissues of each group were collected and used based on two-dimensional electrophoresis(2-DE)-matrix-assisted laser analysis ionization time of flight mass spectrometry(MALDI-TOF-MS)Proteomics analysis methods,The differential expression of wound surface protein between normal rats and agkistrodon rats was compared and analyzed.1.3 Animal experiments to observe the toxic effects of Agkistrodon venom on rats and the therapeutic effects of 717 Jiedu Mixture,in a barrier environment,observe the toxic effects of Agkistrodon venom on rats in terms of organ coefficients,biochemical indicators,and levels of inflammatory factors.And from the NF-κB and MAPK signal pathways to study the anti-Agkistrodon venom mechanisms of 717 Jiedu Mixture.2 Methods2.1 Clinical observation: 150 patients who met the conditions of clinical trial from September 2018 to November 2020 were randomly divided into treatment group and control group,with 75 patients in each group.The control group was given conventional western medicine therapies,while patients in the treatment group take 717 Jiedu mixture orally twice a day(morning and evening after meal daily)in addition to the conventional western therapies.Symptoms of the two groups within 7 days were recorded in detail,Biochemical indicators were detected by biochemical analyzer and TNF-α,IL-6,5-HT,Histamine was detected by ELISA,which are:2.1.1 clinical efficacy.2.1.2 relationship between the degree of illness and clinical efficacy of the two groups.2.1.3 swelling,pain,ecchymosis regression time and cure time.2.1.4 local symptoms,systemic signs score and total score before and after treatment.2.1.5 total score before treatment,1 day,3 days and 7 days after treatment.2.1.6 blood biochemical values such as Myocardial zymogram,liver function indicators,renal function indicators to be tested.2.1.7the expression levels of related factors in two groups before and after treatment.2.2 Animal experiment2.2.1 Experiment 1: 16 SPF-grade SD rats were randomly divided into two groups with half male and female.Agkistrodon venom was subcutaneously injected into the left lower limb of the rat to make a snake wound model.After 2 hours,2 types of wound muscles were taken for protein analysis,2D-DIGE two-dimensional electrophoresis,MALDI-TOF-MS mass spectrometry analysis,observe rat protein expression,use software Mascot distiller to filter baseline peaks and identify signal peaks.Use Mascot software to search the NCBI database to find the matching related proteins,and at the same time to query its functions,to clarify what kind of protein the differential protein is identified.2.2.2 Experiment 2: 52 SPF-grade SD rats(half male and half female)were divided into normal group and Agkistrodon halys venom group.The latter group was further divided into 6 sub-groups,namely2 h,4h,12 h,24h,36 h and 72 h groups.Agkistrodon halys venom was injected into the left hind limb of the rats to establish the model.The heart,liver,lung,kidney tissues and arterial blood were taken respectively at 2h,4h,12 h,24h,36 h and 72 h.Observation:2.2.2.1 reaction of rats after injection of snake venom.2.2.2.2 serum biochemical indexes(CK,LDH,AST,ALT GLDH,ALP,UREA,CREA,UA)were detected by biochemical analyzer.2.2.2.3 pathological changes of organs.2.2.3 Experiment 3: 72 SPF-grade SD rats(half male and half female)were randomly divided into 6 groups: normal control group,model control group,snake venom serum group,low-dose,medium-dose and high-dose group of 717 Jiedu mixture.Agkistrodon halys venom was injected into the left lower limb,and the treatment was given 2 hours later.The snake venom group was injected with 0.6Ml of anti-snake venom serum via tail vein,and the 717 Jiedu mixture group was perfused with 5,20 and 50 mL/kg.d by gavage for6 days.Twenty KM mice were randomly divided into two groups,half male and half female,gavaged with 10 mL/kg.d for 3 days.Liver tissue and blood was taken after abdominal anesthesia at each SD rats group when they were injected with Agkistrodon halys venom at 2 hours,72 hours and 6 days after treatment respectively.Observation:2.2.3.1 The general situation of rats,the peritoneal condition of Agkistrodon viper venom mice,and the liver tissue structure of rats under the microscope.2.2.3.2 the contents of TNF-α and NF-κB in serum were detected by ELISA.the levels of IL-2,IL-4,IL-6,TNF-α,NF-κB,CRP and INF-β in serum were detected on the 6th day.the expression levels of IκBαmRNA、p38MAPK mRNA were detected by RT-PCR,the expressions of iNOS and COX-2 were detected by Western blot.The expressions of NF-кB P50 and NF-кB p65 were detected by immunohistochemistry.3 Results3.1 Clinical research3.1.1 The total effective rates of the treatment group and the control group were 90.41% and 76.39% respectively,and there was significant difference in the clinical efficacy between the two groups(P<0.05).3.1.2 In the treatment group,there were 62 mild and moderate patients,including 17 cured,25 markedly effective,16 effective and 4 ineffective,11 severe patients including 5 cured,2 markedly effective,1 effective and3 ineffective.In the control group,there were 65 mild and moderate patients,8 were cured,20 markedly effective,22 effective and 15 ineffective,7 severe patients,including 4 cured,1 markedly effective,and2 ineffective.There was no significant difference between the two groups of mild and moderate patients(P>0.05),and the difference in the clinical efficacy of severe patients was significant(P < 0.05),which was statistically significant.3.1.3 The average swelling,pain and ecchymosis regression time of the treatment group was 4.48±0.87 days,4.95±0.57 days,7.43±1.42 days,and the average hospitalization time was 4.98±1.17 days,whereas the control group were 5.75±1.01 days,6.50±0.86 days,8.52±1.44 days and 6.46±1.45 days.There were significant differences in swelling,pain,ecchymosis regression and disease cure time between the two groups(P<0.05)with statistical significance.3.1.4 After treatment,the scores of local symptoms,systemic signs and total scores of the treatment group were 1.48±0.87,1.94±1.15 and 2.44±1.44,those of the control group were 2.46±0.86,2.95±1.15 and 4.20±1.30,respectively,with significant differences between the two groups(P<0.01),with statistical significance.3.1.5 717 Jiedu mixture can effectively treat patients with Agkistrodon halys bite.After treatment,the scores of the two groups decreased significantly(P<0.01)on the third day and the seventh day,with statistical significance.3.1.6 There were significant differences in CK,CK-MB,LDH,ALT,AST,BUN and Cr values between the two groups before and after treatment(P<0.05).3.1.7 After treatment,WBC,CRP,APTT,PT,TNF-α,IL-6,5-HT and Histamine values of the two groups decreased,with statistical significance before and after treatment.3.2 Animal experiment3.2.1 Experiment 1Compared with the normal SD rats,the protein expression of Agkistrodon halys rat model increased by 160 spots and decreased by 141 spots.the four spots of agkistrodon’s wound identified proteins mainly containing Myosin-4 and Alpha-1B-glycoprotein.The feasibility of the application of proteomics technology in the screening of early differential diagnosis markers for snake injuries was verified.3.2.2 Experiment 23.2.2.1 In 2 hours,poisoning appeared from the wound.in 4 hours,edema was obvious.in 12 hours,tissue fluid exuded.in 36 hours a trend of self-healing appeared and in 72 hours the wound began to scab.3.2.2.2 In the sterile environment,the organs of Agkistrodon halys venom rats increased or decreased,and each organ had a recovery trend in36 hours.3.2.2.3 After treatment,TP and ALB values in each group decreased.GLOB decreased first and then increased.ALT,AST,ALP,CK,LDH,UREA,CREA,UA values increased first and then decreased.3.2.2.4 Myocardial cells,liver cells and kidney tissue showed obvious injury and inflammatory infiltration at 4h,and tissue dissolution and necrosis at 72 h.In the later stage of lung tissue,there was slight alveolar widening,and inflammatory cell infiltration was rare.3.2.3 Experiment 33.2.3.1 The rats in serum group and 717 Jiedu mixture medium and high dose groups had smooth skin,good mental state,normal diet and activities,and high wound healing degree.The rats in the model group had loose fur,poor mental status,less diet and activity,and slow reaction.After 4 hours of modeling,the abdominal subcutaneous purplish black diffuse bleeding was found in the mice,and the symptoms were relieved 72 hours later,especially in the traditional Chinese medicine group.3.2.3.2 In normal rats,the arrangement of hepatocytes in hepatic lobules was regular and polygonal,In the model group,the structure of hepatic lobules was slightly disordered,the volume of hepatocytes increased,the cytoplasm staining was light,the intracellular vacuolization,there were obvious inflammatory cell infiltration and punctate necrosis.717 Jiedu mixture could reduce the degree of swelling of hepatocytes,especially in the high-dose group,the liver cords were arranged more orderly,and the tissue structure tended to be normal.3.2.3.3 The expression of TNF-α,NF-κB,IL-2,IL-4,IL-6,CRP and INF-βin the serum of rats injured by Agkistrodon halys were increased.COX-2,i NOS,p38 MAPK,p-p38 MAPK,STAT3,P-STAT3,IκBαmRNA,NF-κB,NF-κB P50 protein expression in liver tissue were increased,p38 MAPK mRNA and IκBα protein expression were down-regulated,717 Jiedu Mixture can play an effective reverse regulatory role.4 Conclusion4.1 717 Jiedu mixture can effectively treat patients with Agkistrodon halys bite by shortening swelling,pain,ecchymosis subsiding time and disease healing time,and lowering local symptoms,systemic signs and total scores.4.2CK,CK-MB,LDH,ALT,AST,BUN,Cr,TNF-α,NF-κB,IL-6,p38 MAPK,p-p38 MAP K,STAT3,P-STAT3,IκBαmRNA,NF-κB P50 can be used as the evaluation index of Agkistrodon halys bite degree and curative effect.4.3 717 Jiedu mixture By regulating NF-κB and MAPK signaling pathway have anti Agkistrodon halys venom activity,inhibit the main inflammatory factors,and have anti-inflammatory activity and immunomodulatory effect in the later stage of treatment,which also provides experimental basis for clinical research.