| Dampness heat diarrhea(DHD)is one of the most common syndromes of calf diarrhea.The etiology of this disease is complex,and the mixed infection cases of virus and bacteria are common.At present,this disease mainly depends on antibiotic treatment,so the bacterial drug resistance is prominent.Clinical technology such as Chinese veterinary medicine to replace antibiotics to prevent and treat such diseases is increasingly urgent.The concept of omics is similar to the holistic concept of Traditional Chinese medicine.Omics techniques such as genome and metabolomics have been widely used in the field of Traditional Chinese medicine.Huangbai Jianpi(HBJP)decoction is a new Chinese veterinary medicine compound,which is based on the PD and Si Junzi decoction.It has the effects of clearing heat and detoxifying,eliminating dampness and stopping diarrhea,regulating qi and harmonizing stomach.In this paper,the acute toxicity,therapeutic effect and the in vivo and in vitro mechanism of HBJP decoction were studied respectively,aiming at explaining the mechanism of HBJP decoction in the treatment of DHD in calves,and providing basic data and scientific basis for new drug development and clinical rational application of HBJP decoction.(1)Acute toxicity test of HBJP decoction and evaluation of its therapeutic effect on DHD Acute toxicity test showed that the LD50 of HBJP decoction was more than50000 mg/kg.The maximum dose did not cause death of mice,and had no obvious effect on the clinical signs of mice,and no obvious histopathological changes were observed,indicating that the decoction had no obvious toxic and side effects within a reasonable dose range.The DHD model in rats was established by multiple factors including high-sugar and fat diet,high temperature and dampness environment,biological pathogenic factors.The model rats showed the typical clinical symptoms of DHD,such as short yellow urine,yellow,sticky and smelly feces,and elevated body temperature.In addition,there were abnormal changes in blood routine,blood biochemical indexes and histopathological diagnosis results of rats in model group.After being treated with HBJP decoction,the clinical symptoms in rats were alleviated,the pathological changes of liver,lung and intestine were improved,body weight increase,body temperature,blood biochemical and blood routine indexes also returned to normal levels.According to the theory of Traditional Chinese veterinarian,the diagnostic criteria of clinical cases of calves with DHD were established,and 46 natural cases of DHD were included.Rotavirus,coronavirus,cryptosporidium,Escherichia coli K99 and giardia were detected in the collected anal swabs,among which the proportion of mixed infection is 39.13% and the detection rate of coronavirus is the highest(34.78%).The levels of uric acid,urea nitrogen and creatinine in serum of sick calves were abnormal.The levels of cytokines IL-10 and TFF3 were significantly decreased,and the levels of IL-1β were significantly increased in the serum of diseased calves.After treatment with HBJP decoction,the clinical symptoms of the sick cattle were significantly reduced or even completely recovered,the effective rate was84.78%,and the blood biochemical indexes and related cytokines levels were basically restored to normal after treatment.(2)To explore the pathogenesis of calves with DHD based on omics techniques16 S r DNA sequencing showed that compared with the results of healthy calves,the microbial diversity including Escherichia-shigella,enterococcus and Bacteroides changed significantly in calves with DHD.Proteomic results showed that 52 proteins including the haptoglobin precursor,xanthine dehydrogenase and coagulation factor XIII B(F13B)were significantly changed in calves with DHD.These proteins are involved in a wide range of biological processes such as purine metabolism,intestinal immune network for Ig A production and PI3 K signal pathway.The 13 differential proteins were screened for parallel response monitoring.The results showed that,fibronectin precursor and apolipoprotein C-IV precursor were closely related to calves with DHD and could be used as biomarkers for calves with DHD.The results of metabolomics showed that 194 metabolites,including choline,phosphatidylcholine and lecithin,related to alpha-Linolenic acid metabolism,linoleic acid metabolism and glycerophospholipid metabolism,were significantly differential in plasma between calves with DHD and healthy calves.(3)Study on the mechanism of HBJP decoction in treating calves with DHD Proteomic results showed that the polymeric immunoglobulin receptor and alpha-1-acid glycoprotein precursor were significantly down-regulated,while F13 B and hemoglobin subunit beta were significantly up-regulated after treatment with HBJP decoction.These proteins were significantly associated with complement and coagulation cascades,intestinal immune network for Ig A production,and acute inflammatory response.Metabolomics results showed that 300 metabolites,such as choline,methionine and pyripropanol,were significantly changed after treatment with HBJP decoction.They were associated with 53 signaling pathways,of which 9 pathways were significantly enriched,including alpha-linolenic acid metabolism,linoleic acid metabolism and arachidonic acid metabolism.The results of 16 S r DNA gene sequence detection showed that the levels of intestinal microbes such as megasphaera,bifidobacterium and phascolarctobacterium were significantly changed after treatment.The proteins levels of P-PI3 K,P-Akt and P-NF-κB related to PI3K/AKT/NF-κB signal pathway were significantly increased in colon of DHD rats,and the expression levels of IL-1β,IL-2 and IL-6 in plasma were significantly increased,while the expression levels of IL-10 were significantly decreased.After treatment with HBJP decoction,the phosphorylation level of these proteins decreased,the expression level of pro-inflammatory cytokines was decreased,and the expression level of anti-inflammatory cytokines was increased.These indicators were further verified in a model of inflammation by TNF-α-stimulated of IEC-6.These results suggest that HBJP decoction can reduce intestinal inflammatory response by inhibiting PI3K/AKT/NF-κB signaling pathway.In conclusion,HBJP decoction can regulate the disordered metabolism of alphalinolenic acid,linoleic acid and glycerophospholipid,and regulate the abnormal expression of XIII B and IGLV1-40,and regulate the intestinal immune network for Ig A production,and improve intestinal microbial balance related to purine,pyrimidine and amino acid metabolism,interfere with PI3K/AKT/NF-κB signal pathway to inhibit inflammation,and thus play a role in the treatment of calves with DHD.HBJP decoction is an effective candidate drug for the treatment of calves with DHD,and has further development value. |
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