Screening Of Anti-inflammatory Constituents In Polygala Japonica Houtt. To Repair The Malassezia-stimulated Skin Injury

Objective:Chinese traditional medicine Polygala japonica Houtt.(PJ),a member of Polygala L.family,has the effects of detoxification and pain relief.PJ is often used to treat upper respiratory tract infections such as tonsillitis and cough,which shows the potential of anti-inflammatory.In this experiment,the lipopolysaccharide(LPS)-induced RAW264.7macrophage inflammation model was used to screen the anti-inflammatory active components of PJ,and the anti-inflammatory mechanism was preliminarily explored at the molecular level.On this basis,an in vitro Malassezia-infected skin of the C57BL/6 mouse model was used to verify its anti-inflammatory effect,providing a theoretical scientific basis and explanation for the development of PJ topical preparations.Methods:(1)MTT method was used to detect cell viability.LPS-induced RAW264.7 macrophage inflammation model was used to screen the anti-inflammatory active components of PJ.(2)Optical microscope was used to observe cell morphology changes.An assay kit was used to determine the level of nitric oxide(NO)and intracellular reactive oxygen species(ROS)to analyze the anti-inflammatory effect.Quantibody array was used to detect the content of 40 extracellular inflammatory cytokines.The levels of intracellular and extracellular inflammation-related cytokines(IL-1β,IL-6,IL-18,TNF-α)were measured through real-time quantitative polymerase chain reaction(RT-qPCR).The possible mechanism of Polygalaxanthone Ⅲ(POL)was analyzed by the Kyoto Encyclopedia of Genes and Genomes(KEGG)and verified by Western Blot(WB).(3)Preparation of medicine cream by emulsification method.Use an electric scrub roller to slightly rub the back skin of C57BL/6 mice,and apply Malassezia olive oil suspension to the wound surface to prepare a Malassezia-induced skin infection model.Mice were randomly divided into 6 groups: normal group,skin injury group,model group,blank cream group,PJ group,POL low-dose group(1% POL),and POL high-dose group(2% POL).From the day of modeling,observe the physiological activities and weight changes of the mice and take pictures to record the wound healing.On the 6th and 12 th day of the administration,half of the mice in each group randomly chose to sacrifice.The injured tissue was collected and take bacteria inspection and sampling.Hematoxylin-eosin(HE)and Masson staining were used to observe the morphology of skin wound tissue.WB was used to detect protein expression in skin tissue homogenate.(4)SDS-PAGE was used to analyze the protein expression of Malassezia bacterium supernatant.A bacteriostatic circle experiment was used to investigate the relationship between POL and Malassezia.Malassezia bacterium supernatant was used to stimulate HaCat cells and detect the effect of POL on lipid secretion.Results:(1)Compared with other components of PJ(Polygalasaponin XLIX PS-(XLIX),Polygalasaponin E(PS-E),and Polygalasaponin F(PS-F)),POL(within the safe dose range,<2.0 mM)could reduce the NO content in the RAW264.7 cell model induced by LPS and be concentration-dependent,showing best anti-inflammatory activity.(2)POL significantly improves the morphological changes of RAW264.7 cells induced by LPS,reduces pseudopodia and cell vacuoles,reduces the effects of LPS on cell viability,ROS and iNOS,and down-regulated IL-1β,IL-6,IL-18,and TNF-α gene expression(p<0.001,vs.LPS induction group).(3)Compared with the LPS treatment group alone,POL significantly reduced the 10 pro-inflammatory factors(IL-21,GM-CSF,ICAM-1,IL-15,TARC,PF4,TCA-3,IL-3,IL-17 A,and KC),promoted the secretion of four anti-inflammatory factors(IL-10,G-CSF,IL-2,and TIMP-1),and have a stronger regulatory effect on pro-inflammatory factors.The inhibition rate of IL-21 and GM-CSF by high-dose POL(2.0 mM)pretreatment reached 72.0±5.4% and54.1±4.8%,respectively).(4)KEGG pathway enrichment analysis predicted that POL may exert its anti-inflammatory effects through the JAK-STAT signaling pathway.WB verification results showed that POL pretreatment could significantly inhibit the phosphorylation of JAK1,JAK2,and STAT1(p<0.001,vs.LPS induction group).(5)Successfully established a mouse skin injury model of Malassezia infection.Compared with simple skin injury,the skin injury combined with Malassezia can significantly aggravate the inflammation of the wound,increase the injury degree,and prolong the wound healing time.Compared with the model group,the blank cream,PJ cream,and POL cream could all reduce the injury degree and shorten the wound healing time.The wounds of the POL high-dose cream group had all healed on the 7th day.In addition,the existence of Malassezia was not detected in the wounds of the simple skin injury group and the POL cream group,while Malassezia was still detected in the other groups on the 6th day.(6)The POL treatment group significantly reduced the amount of lipid produced by the HaCat cells stimulated by the supernatant of Malassezia bacteria liquid,but it did not directly inhibit the growth of Malassezia.Conclusion:(1)The RAW264.7 cell inflammation model induced by LPS was used to screen out the natural anti-inflammatory active compound POL from PJ.POL significantly reduces the inflammatory factors mainly through the JAK-STAT signaling pathway.(2)POL cream can treat skin damage caused by Malassezia infection,reduce wound inflammation and shorten healing time.POL cannot directly inhibit the growth of Malassezia,but it can significantly reduce the number of lipid droplets in HaCat cells stimulated by Malassezia.