Study On The Anti-inflammatory And Analgesic Effects Of Kaempferol And Its Mechanism

Inflammation is one of the body’s defense responses to external harmful stimuli.Inflammation is very common and can occur in various tissues and organs of the body.Clinical manifestations of inflammation include redness,swelling,heat,pain and other symptoms.Inflammatory pain is a normal reaction in our body,it occurrs when the inflammatory factor is being cleared.It can cause the body to produce physiological discomfort,it can also affect the body’s normal physiological and psychological health if the symptoms aggravated.Clinically,the most common Drugs for the treatment of inflammatory pain are Paracetamol,Aspirin(2-(Acetoxybenzoic Acid),also known as Acetylsalicylic Acid)and other Nonsteroidal Antiinflammatory Drugs(NSAIDs),but they all have some side effect,such as gastrointestinal discomfort or liver and kidney function damage.Therefore,the author focused on finding new anti-inflammatory and analgesic drugs with less side effects and better anti-inflammatory and analgesic effects.Kaempferol(KF)is a kind of natural edible flavonoids,which is mainly derived from the rhizome of the Zingiberaceae plant Kaempferol.It is widely found in various vegetables and fruits,and has many effects such as anti-cancer,anti-oxidation,anti-inflammatory,ect.In view of its anti-inflammatory effect,the author wanted to explore whether kaempferol has a good analgesic effect on inflammatory pain and its anti-inflammatory analgesic mechanism,so as to provide a new idea for the clinical treatment of inflammatory pain.Objective:1.Animal behavior experiment was used to investigate whether kaempferol,an extract from rhizome of kaempferol,has anti-inflammatory and analgesic effects.2.Explore the anti-inflammatory and analgesic effects of kaempferol and its mechanism from the individual,cellular,and mRNA levels of animals by using animal behavior and immunohistochemistry techniques.Experimental Methods:1.C57BL/6 mice at 6-8 weeks were randomly divided into WT control group,CFA model group(CFA 25 μL/mouse)according to body weight,kaempferol high-dose group(50 mg/kg/Day),kaempferol low-dose group(30 mg/kg/Day)and Diclofenac Sodium group(10 mg/kg/Day).In each group n=6.At the odd and the even days after modeling,the sensitivity of mice to mechanical stimulation and thermal stimulation was measured separately,and the change curves were recorded by 50%paw withdrawal threshold and thermal pain latencys.2.After 14 days of modeling,cDNA was extracted from L4-L6 DRG segments of mice in WT control group,CFA model group and KF administration group(50 mg/kg/Day),and then reverse transcripted into RNA.mRNA expression of pain-related channels and related inflammatory factors was measured by RT-qPCR.3.After 14 days of modeling,L4-L6 DRG segments of mice from WT control group,CFA model group and KF administration group(50 mg/kg/Day)were taken out to culture primary cells.The sensitivity of mouse DRG neurons to β-alanine(1-4 mmol/L)at different concentrations of MrgprD was detected by Ca2+ imaging technique.4.Male C57BL/6 WT mice about 6-8 weeks old were randomly divided into control group(n=6)and kaempferol group(n=6).The 48℃ water bate tail flick time baseline were recorded before medicine intragastric administration.Then the mice in each group were respectively given kaempferol(50 mg/kg)and saline(0.9%NaCI,0.1 ml)by intragastric administration to test the tail flick time curve by immerse about 1/3 tail of the mice into the water after 30,60,90,120.180 minutes separately.5.Male mice about 6-8 weeks old were randomly divided into WT control group and KF administration group(50 mg/kg).After one hour of kaempferol intragastric administration,the pain was induced by subcutaneous injection of 10μl 500μM capsincine in the right posterior foot.The times of paw lifting and paw licking time were observed and recorded.6.Male mice about 6-8 weeks old were randomly divided into WT control group and KF administration group(50 mg/kg).After one hour of kaempferol intragastric administration,the pain was induced by subcutaneous injection of 1%carrageenan(20μl)in the right posterior foot to induce pain.The Paw Withdrawal Threshold was tested after 1,3,5h to form the change curve.7.Male mice about 6-8 weeks old were randomly divided into WT control group and KF administration group(50 mg/kg).After one hour of kaempferol intragastric administration,the pain was induced by subcutaneous injection of 25μl CFA in the right posterior foot.The times of paw lifting and paw licking time were observed and recorded.Hematoxylin-eosin(HE)staining technology was used to observe the aggregation of skin inflammatory factors in the foot modeling site of the WT control group,CFA modeling group and high-dose administration group(50 mg/kg/Day)to evaluate the anti-inflammatory effect of kaempferol.9.Male MrgprD-/-mice and wild-type homologous C57BL/6 mice were selected to observe the behavioral changes of mice after CFA modeling and KF administration(50 mg/kg/Day)through mechanical and thermal radiation stimulation,so as to evaluate the role of MrgprD channel in CFA model and the therapeutic effect of KF.Experimental results:1.Kaempferol had no effect on normal mice and had no obvious toxic and side effects.2.Kaempferol can effectively relieve acute and chronic inflammatory pain in mice,improve the inflammation of the plantar modeling site,and reduce the aggregation of inflammatory factors.After modeling,the sensitivity of KF administration group mice to mechanical and thermal stimulation is reduced,but it is no use to acute noninflammatory pain.3.Kaempferol can effectively reduce the mRNA expression of IL-6,TNF-α,IL-1β,IL-8,MrgprD,TRPA1,TRPV1,and Nav 1.8 induced by CFA modeling,but has no significant effect on the expression of TRPV4 and Myd88.4.Kempferol administration inhibited β-alanine induced calcium influx in mouse DRG neurons and HEK293 cells.Kaempferol has a good anti-inflammatory and analgesic effect on CFA-induced inflammatory pain,and this analgesic effect may be related to the decreased expression of MrgprD.