Anti-inflammatory Effect Of Hedyotis Diffusa And High-throughput Sequencing Of MiRNA

Objective:Exploring the therapeutic effect of Hedyotis diffusa on inflammation;To explore the possible anti-inflammatory mechanism of Hedyotis diffusa in innate immunity,and provide experimental basis for clinical treatment;Identification and analysis of known microRNAs in Hedyotis diffusa and prediction of new microRNAs in Hedyotis diffusa will provide a basis for further research on the anti-inflammatory mechanism of the expression of microRNAs and target genes in Hedyotis diffusa?Methods:Xylene induced mouse ear swelling model.Kunming mice were randomly divided into a blank group,a positive control group?aspirin?,a low,medium,and high dose of Hedyotis diffusa extract,low,medium,and high doses of Hedyotis diffusa extract.Continuous tube feeding for 7 days,1 hour after the last administration,30?l of 100%xylene was applied to both sides of the right ear of the mouse,and the left ear was compared.After 30 minutes,The anti-inflammatory effect of Hedyotis diffusa was studied by cutting the left and right ears along the auricle baseline and making ear slices with a perforator?radius 4 mm?.SD rats were randomly divided into model group,positive control group?aspirin?,low,medium and high dose groups of water extract of Hedyotis diffusa,low,medium and high dose groups of alcohol extract of Hedyotis diffusa,to ensure aseptic conditions.After chloral hydrate anesthesia,abdominal incision was made and weighed cotton balls were implanted subcutaneously in both inguinal and inguinal regions of rats.The rats were sacrificed after the last Administration for 12 days.Cotton ball granulation tissue was completely peeled off and dried in oven at75?for 24 hour.The weight of the original cotton ball was reduced,which was the net weight of granuloma.The weight of granulation was compared and the inhibition rate was calculated.The LD₅₀0 assay was first performed using the“koushifa”^[1],then a sepsis model was established.After weighing,mice in the model group were injected with LD50 dose LPS?0.2ml/10g?intraperitoneally,and the negative control group and the traditional Chinese medicine control group were given the same amount of normal saline.Mice were randomly divided into blank control group,model group,dexamethasone intervention group and Hedyotis diffusa water extraction group,alcohol extraction group.The dexamethasone intervention group,the water extraction group and the alcohol extraction group were continuously intragastrically administered for 7 days,and the other groups were given an equal volume of normal saline.After the last administration for 30minutes,except for the blank group,the other groups of mice were intraperitoneally injected with LPS?10mg/kg?.In the model group,the blank group was given an equal volume of physiological saline,once per day until the end of the experiment.The clinical symptoms were observed at 6h,12h,24h and 48h after model establishment.The serum inflammatory factors interleukin-6?IL-6?,interleukin-1??IL-1??and tumor were detected by enzyme-linked immunosorbent assay?ELISA?.Necrosis factor-alpha?TNF-alpha?levels.The mice were sacrificed by cervical dislocation.The mice were sacrificed by cervical dislocation.The histopathological changes of the liver were observed.The left upper lobe of the liver was cut and fixed in 10%neutral formalin solution.The paraffin sections were made into 5 micron thick sections.The histopathological changes of the liver were observed under light microscopy.The total RNA of Hedyotis diffusa Willd was extracted by Trizol,and the purity and integrity of RNA were detected by agarose gel electrophoresis,TGem micro spectrophotometer and Agient 2100Bioanalyzer.Then the smallRNA Library of Ophiopogon diffusa was constructed.Using Unigene as the reference sequence,Clean read was compared with Gene,Rfam,microRNA Base 21.0 and Repbase databases,and the known microRNAs and their expression levels were obtained.The unmatched sequences were used for the prediction and analysis of new microRNAs.Results:The ear swelling experiment showed that the aqueous extract and ethanol extract of Hedyotis diffusa had obvious inhibitory effect on xylene-induced ear swelling in mice.Among them,the high-dose group of water extracts,the low-dose group and the middle-dose group of alcohol extracts have obvious inhibitory effects.The granuloma experiment results showed that the high-dose group of Hedyotis diffusa water extract;the low-dose and medium-dose alcohol extracts significantly inhibited the granuloma caused by cotton balls.The results of LPS sepsis test showed that the degree of liver tissue damage in the mice of the Hedyotis diffusa extract group was significantly lighter than that in the model group,and the damage in the water extraction group was more obvious;Compared with the model group at the same time,the levels of serum IL-6,IL-1?and TNF-?in the mice treated with Hedyotis diffusa aqueous extract and Hedyotis diffusa alcohol extract were significantly decreased?P<0.05?.In the comparison between groups,the above indicators of the 12h node of Hedyotis diffusaextract group were better than the 6h node,and compared with the Hedyotis diffusa ethanol extract group,the Hedyotis diffusa aqueous extract group at each time point Better results but no significant differences.Gel electrophoresis results showed that the total 18S and 28S bands of the total RNA of Hedyotis diffusa Willd were clear and complete,and the brightness ratio was close to 2.The results of Agient 2100Bioanalyzer quality test showed that the OD260nm/OD230nm value was1.8-2.1,and OD260nm/OD280nm ratio was 2.0-2.3,28S/18S>1.0 and RIN>7.0,which met the standard of high throughput library building.Through high throughput sequencing,115 known microRNAs were identified in Hedyotis diffusa,belonging to 16 microRNAs.The highest expression levels of microRNAs such as micro166,micro157,micro2911,micro167 and micro168 were all over 5000.Conclusion:The extract of Hedyotis diffusa can effectively inhibit the swelling of mouse ears and the granuloma caused by cotton balls.When the concentration of the aqueous extract was 3g/kg,the inhibition was most pronounced.When the alcohol extract concentration is125mg/kg,the inhibition is the strongest.Hedyotis diffusa extract can effectively improve liver damage in mice with LPS sepsis and alleviate the clinical symptoms of mice;it can effectively inhibit the levels of IL-6,IL-1?and TNF-?,and its mechanism may be through inhibition The expression of related inflammatory factors plays a role in regulating the balance of the innate immune system.The total RNA of Hedyotis diffusa was successfully extracted and the smallRNA library was constructed.The species and expression level of microRNA in Hedyotis diffusa were identified.It was confirmed that fresh Hedyotis diffusa was rich in microRNA.