| Mastitis is an acute or chronic inflammatory disease caused when breast tissue is infected by pathogenic microorganisms through the nipples.The main characteristics of the disease are the physical and chemical properties of milk and the pathological changes of breast tissue,which has led to a significant reduction in milk production and the quality of dairy products.As far as dairy cows are concerned,the economic losses caused by mastitis worldwide are as high as$ 40 billion per year.For many years,the main treatment ideas and methods for treating mastitis are still antibiotics.Although antibiotics can effectively kill pathogenic microorganisms in the early stage,they cannot effectively continue to cause inflammation.On the other hand,with the continuous improvement of people’s requirements for food safety and stricter national requirements for the quality of dairy products,the negative effects of antibiotic treatment have to be worrying.Over time,the abuse of antibiotics has led to the generation of pathogenic bacteria.Drug resistance and drug residues in the body are endangering the health of the body..Therefore,it is of great significance to find new prevention and treatment drugs for mastitis.However,Chinese herbal medicine has played a great advantage in low toxicity,low drug residues and non-drug resistance,and is increasingly sought after by scientific researchers.Forsythia as a commonly used Chinese medicine has a wide range of pharmacological activities.It has very strong antibacterial and anti-inflammatory effects.Forsythiaside A is one of the main active ingredients isolated from the dried fruits of Forsythia suspensa and is reported to have anti-inflammatory and antioxidant effects.LPS(lipopolysaccharide)is a thicker lipopolysaccharide substance in the outermost layer of the cell wall of Gram-negative bacteria.It is one of the strongest causative factors that cause mastitis.Gram-negative bacteria such as E.coli enter the mammary gland through the milk duct to multiply,and then the bacteria die and lyse to release a large amount of LPS under the action of lysozyme in animals.Cells and tissues cause damage,which stimulates the body to produce an inflammatory response,produces a variety of inflammatory factors and mediators, and induces the activation of a variety of inflammatory signaling pathways.In this experiment,a mouse mastitis model was established by injecting mouse mammary glands with LPS to investigate the prevention and treatment mechanism of forsythiaside A on mastitis.The p38 mitogen-activated protein kinase(p38MAPK)signaling pathway and its downstream nuclear factor-κB(NF-κB)are considered to be classic pathways of the inflammatory response.In this study,the expression levels of inflammatory cytokines and pathological changes in breast tissues of mice with mastitis induced by LPS were examined.It was also clarified that forsythin reduced LPS-induced mastitis in mice by inhibiting NF-κB and p38 MAPK signaling pathways.This test is based on the dialectical analysis of mastitis by traditional Chinese medicine.Forsythia,which has the functions of clearing heat and detoxifying,swelling and dispersing,and evacuating wind and heat,is selected as the research drug.The classic clearing and detoxifying traditional Chinese medicine dandelion and violaceum are used as the control.The mouse is an animal model,and the monomer components with high anti-inflammatory activity in Forsythia suspensa were screened,and the mechanism of its action in the prevention and treatment of mastitis was explored.This test is divided into three parts:1.Anti inflammatory activity of Forsythia suspensa extractThe anti-inflammatory effect of Forsythia suspensa extract was investigated by ear swelling induced by xylene and peritoneal capillary permeability test induced by acetic acid.High(1.6 g/kg),medium(0.8g /kg)and low(0.4 g/kg)alcohol extract groups were set up,with Viola,dandelion and dexamethasone as drug control,and inflammatory model group and blank control group were set up.After oral administration,the inhibition rate of xylene induced ear swelling and OD value of capillary permeability exudation dye induced by acetic acid were calculated and detected,and the anti-inflammatory effect of each experimental drug group was statistically analyzed.The results showed that the anti-inflammatory effect of Forsythia suspensa was the best when the concentration was 1.6 g/kg.2.Study on Anti-inflammatory Activity of Forsythia Suspense in Different Polar Parts.In order to screen the anti-inflammatory components in Forsythia suspensa,different polar segments were extracted from Forsythia suspensa,and the antiinflammatory activities of the extracts were compared.First of all,the essential oil was extracted from Forsythia suspensa by boiling and distilling,then the aqueous phase extract was filtered and recycled for use.The residue was extracted by heat reflux with 75% ethanol.The extract was filtered,and the two filtrates were combined with the above water extract to obtain the whole component extract of Forsythia suspensa.Then the extract was concentrated under reduced pressure and then extracted with equal volume of ethyl acetate.The extract was concentrated and dried under reduced pressure to obtain the ethyl acetate extract of Forsythia suspensa.After ethyl acetate extraction,the solution was added with equal volume of water to saturate n-butanol,and then extracted and concentrated to obtain nbutanol extract.The aqueous extract of Forsythia suspensa was obtained by decompressing the remaining aqueous solution.The anti-inflammatory activity of extracts from Forsythia suspensa with different polarity was observed by xylene induced ear swelling test and acetic acid induced peritoneal capillary permeability test.The results showed that in xylene induced ear swelling and acetic acid induced peritoneal capillary permeability test,the extracts of ethyl acetate and n-butanol of Forsythia suspensa were significantly different from those of the model group(P<0.05),and the anti-inflammatory activity was significant;the volatile oil of Forsythia suspensa was 0.8 g/kg and 1.6 g/kg,respectively The anti-inflammatory activity of the water extract was significant(P<0.05),but it was not significant compared with the model group(P>0.05).Comprehensive analysis showed that the n-butanol extract of Forsythia suspensa had the highest anti-inflammatory activity.3.Effects of forsythiaside A on NF-κB and p38 MAPK signaling pathway in mouse mammary gland inflammation model.Forsythiaside A is the main biologically active ingredient extracted from forsythia,and has a variety of biological properties,especially anti-inflammatory properties.In this study,the protective effect of forsythiaside A in mice with acute mastitis induced by lipopolysaccharide(LPS)was investigated.A mouse mastitis model was established by LPS milk duct injection.Postpartum female rats were divided into forsythin A low dose(40 mg/kg)+ LPS group,medium dose(80 mg/kg)+ LPS group,high dose group(160 mg/kg)+ LPS,blank control group and LPS model control group.Forsythia suspensa group A was administered continuously for 1week,and both the blank and model rats were given normal saline.After the last dose,the fourth pair of breasts of mice in the forsythia A group and the model control group was injected with LPS to induce breast inflammation in the mice.The blank group was injected with the same amount of physiological saline.Breast tissue was collected at 6h,12 h,24h,and 48 h after inflammation.QPCR was used to detect the m RNA expression levels of IL-1β,IL-6,TNF-α,p38 MAPK,IκBα,and NF-κB.Pathological changes of the breast tissue were observed by HE staining.The contents of IL-1β,IL-6 and TNF-α inflammatory factors were detected by ELISA.Western blot analysis of p38 MAPK,p-p38 MAPK,p-p65NF-κB,p65NF-κB,and IκBα protein expression levels in breast tissue.The results showed that the expression of IL-1β,IL-6,TNF-α,p38 MAPK,IκBα and NF-κB were up-regulated in the LPS model group,and obvious pathological changes were observed in the sections.After forsythiaside A intervention,compared with the LPS model group,the expression levels of the above factors were significantly reduced,and the inflammation symptoms were also significantly reduced by section observation.It is indicated that forsythiaside A can effectively inhibit LPS-induced mammary inflammation in mice by regulating the NF-κB and p38 MAPK signaling pathways.Forsythiaside A in the middle dose group(80mg / kg)has the most significant anti-inflammatory activity. |
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