| BackgroundCelastrol(CEL)is a quinone methyl triterpenoid compound extracted from the rhizomes of tripterygium wilfordii,which has shown good effects in improving inflammatory diseases,tumor diseases,metabolic diseases and neurodegenerative diseases.In addition,CEL can also play an analgesic role in chronic inflammatory pain in animals,and has attracted considerable attention from researchers.The establishment of experimental pain model is the basis for the study of injury perception and pain.Formalin induced inflammatory pain model is a commonly used acute inflammatory pain mouse model,which can generate sustained rather than transient pain stimulation and response by subcutaneously injecting a certain dose of formalin solution into the dorsal side of the right hind foot of mice.The continuous rather than transient pain stimuli and responses generated by this model are reproducible and quantifiable,and are widely used in the evaluation of analgesic effects of different drugs.This project aims to test the improvement effect of CEL on acute inflammatory pain by establishing a mouse model of formalin induced inflammatory pain.q RT-PCR,Western blot,ELISA,immunohistochemical staining and immunohistochemical fluorescence staining were used to explore the possible mechanism of CEL’s analgesic effect in formalin-induced inflammatory pain model mice.ObjectiveTo investigate the analgesic effect and mechanism of CEL on formalin induced inflammatory pain in mice.Method1.Establishment of formalin induced inflammatory pain model miceThe mice were subcutaneously injected with 2.0% formalin solution 20 μL on the dorsal side of the right hind foot to produce a sustained but not temporary pain stimulus and response,and then the mice produced the pain behavior of licking/biting the right hind paw.2.Pain behavior experimentMice with formalin induced inflammatory pain were injected intraperitoneally with CEL,Morphine,NXL or the same volume of solvent.Behavioral tests were conducted 30 minutes later to determine the effects of these treatments on pain behavior of formalin induced inflammatory pain mice.(1)To observe the effect of CEL on the nociceptive behavior of formalin induced inflammatory pain mice by intraperitoneal injection.The experimental animals were divided into 6 groups: control group;CEL group(0.05,0.1,0.5,1.0 mg/kg);Morphine group(0.5 mg/kg);(2)The effect of CEL on the nociceptive behavior of formalin induced inflammatory pain mice was detected by intragastric administration.The experimental animals were divided into 6 groups: control group;CEL group(0.05,0.1,0.5,1.0 mg/kg);Morphine group(0.5 mg/kg);(3)The effect of opioid receptor antagonist NXL on CEL-induced analgesia was tested by intraperitoneal injection.The experimental animals were divided into 4 groups: control group;CEL group(1.0 mg/kg);NXL group(1.0 mg/kg);CEL(1.0 mg/kg)+NXL(1.0 mg/kg)combined injection group.3.The effects of CEL(0.05,0.1,0.5,1.0 mg/kg)on the autonomic motor function of mice were detected.On the third day,after intraperitoneal injection of CEL for 30 min,the mice were immediately placed into the mouse autonomous activity instrument,which had five identical autonomous activity boxes,and the numbers were recorded and put into them respectively.The cumulative number of activities of the mice within 30 min was recorded in the unit of 5 min.4.The effects of CEL(0.05,0.1,0.5,1.0 mg/kg)on motor coordination in mice were detected.On the third day,after CEL was intraperitoneally injected for 30 min,mice were immediately placed on the rod rotator for 6 min,with initial speed of 20 lap/min,primary speed of 30 lap/min and acceleration time of 60 s,so as to observe the effect of CEL on motor coordination function of mice.5.q RT-PCR was used to detect the m RNA expression levels of related genes in different brain tissues and spinal cord tissues(L4-6)of mice with formalin induced inflammatory pain.30 min after intraperitoneal injection of CEL or vehicle,20 μL 2% formalin solution was injected subcutaneously into the dorsal right hind paw.30 min later,the mice were anesthetized,and brain and spinal cord tissues(L4-6)were collected.Before the experiment,the tissues were melted on ice,homogenized,and RNA was extracted.The results were detected by q RT-PCR Penk、Pomc、Pdyn、Dor、Mor、Kor、Nfkb、Nf-kb1、Nf-kb2、Bdnf、Nos2、Nr1、Nr2A、Nr2B、Erk1、Erk2、Jnk、P38、c-Fos、Creb、Egr1and Jun Expression levels of m RNA.6.Western blot was used to detect the expression of related proteins in the spinal cord tissue(L4-6)of mice with formalin induced inflammatory pain.30 min after intraperitoneal injection of CEL or vehicle,20 μL 2% formalin solution was injected subcutaneously into the dorsal side of the right hind paw.30 min later,the mice were anesthetized,and the spinal cord(L4-6)of the mice was collected,flash frozen in liquid nitrogen and stored in the refrigerator at-80℃.Before the experiment,the tissues were thawed on ice,homogenized,and proteins were extracted,and the protein content of KOR,CAMKII,p-CAMKII,ERK,p-ERK,CREB,and p-CREB in the spinal cord was detected by Western blot.7.The levels of dynorphin(Dyn)in serum and spinal cord tissue(L4-6)of mice with formalin induced inflammatory pain were detected by ELISA.30 min after intraperitoneal injection of CEL or vehicle,20 μL 2% formalin solution was injected subcutaneously into the dorsal right hind paw of the mice.30 min later,the mice were anesthetized and blood samples were collected from the eyes.Mouse spinal cord tissues(L4-6)were dissected,homogenized,and the supernatant was collected.Dynorphin(Dyn)content in serum and spinal cord tissue(L4-6)was measured by mouse Dyn ELISA kit.8.The number of c-Fos positive cells in the spinal cord tissue(L4-6)of mice with formalin induced inflammatory pain was detected by IHC30 min after intraperitoneal injection of CEL or vehicle,20 μL 2% formalin solution was injected subcutaneously into the dorsal side of the right hind paw.30 min later,the mice were anesthetized and then perfused into the heart.The number of c-Fos positive cells in the spinal cord(L4-6)was detected by IHC.9.IF was used to detect the number of astrocytes(GFAP)positive cells in the spinal cord tissue(L4-6)of mice with formalin induced inflammatory pain.30 min after intraperitoneal injection of CEL or vehicle,20 μL 2% formalin solution was injected subcutaneously into the dorsal side of the right hind paw.30 min later,the mice were anesthetized and then perfused into the heart.The number of astrocytes in the spinal cord(L4-6)was measured by IF.Results1.Intraperitoneal injection of CEL significantly reduced the time spent licking/biting the right hind paw in mice with formalin induced inflammatory pain.(1)Compared with the control group,at the first phase(0-10 min),intraperitoneal injection of CEL at 0.05,0.1,0.5,1.0 mg/kg did not significantly change the licking/biting time of the right hind paw in mice with formalin induced inflammatory pain.(2)Compared with the control group,intraperitoneal injection of CEL at doses of 0.5 mg/kg and1.0 mg/kg significantly reduced the time spent licking/biting the right hind paw of mice with formalin induced inflammatory pain at the second phase(10-30 min).2.Oral administration of CEL significantly reduced the time spent licking/biting the right hind paw in mice with formalin induced inflammatory pain.(1)Compared with the control group,at the first phase(0-10 min),when the doses of 0.05,0.1,0.5,and 1.0 mg/kg CEL were given by gavage,there was no significant change in the licking/biting time of the right hind paw of mice with formalin induced inflammatory pain.(2)Compared with the control group,intragastric administration of CEL at doses of 0.1,0.5,and1.0 mg/kg significantly reduced the time spent licking/biting the right hind paw of mice with formalin induced inflammatory pain at the second phase(10-30 min).3.Cel-induced analgesia was blocked by the opioid receptor antagonist NXL.Compared with the control group,injection of NXL(1.0 mg/kg)had no effect on the pain behavior of mice in the I and II phases of formalin induced inflammatory pain model.After co-administration of NXL(1.0 mg/kg)and CEL(1.0 mg/kg),the analgesic effect of CEL was significantly blocked by NXL in phase II.4.Behavioral results showed that intraperitoneal injection of CEL(1.0 mg/kg)did not affect the motor coordination function and autonomic activity function of mice with formalin induced inflammatory pain.5.q RT-PCR results showed that compared with the control group,the expression of Pdyn and Kor m RNA in the spinal cord tissue(L4-6)of mice with formalin induced inflammatory pain was significantly increased after intraperitoneal injection of CEL(1.0 mg/kg),and the expression of c-Fos and CREB m RNA was decreased.6.Western blot results showed that the expression levels of KOR,CAMKII and ERK proteins in the spinal cord tissues(L4-6)of mice with formalin induced inflammatory pain were significantly increased after intraperitoneal injection of CEL(1.0 mg/kg)compared with the control group.The expression levels of p-CAMKII,p-ERK and p-CREB proteins were significantly down-regulated.7.ELISA results showed that the expression of dynorphin(Dyn)in the spinal cord tissue(L4-6)of mice with formalin induced inflammatory pain was significantly increased after intraperitoneal injection of CEL(1.0 mg/kg)as compared with the control group.8.The results of IHC showed that compared with the control group,the number of c-Fos positive cells in the spinal cord tissues(L4-6)of mice with formalin induced inflammatory pain was significantly increased after intraperitoneal injection of CEL(1.0 mg/kg).9.The results of IF showed that the activation of astrocytes in the spinal cord tissues(L4-6)of mice with formalin induced inflammatory pain was inhibited after intraperitoneal injection of CEL(1.0 mg/kg)compared with the control group.Conclusion1.Peripheral injection of CEL has analgesic effect in formalin induced inflammatory pain model mice.2.Peripheral CEL injection did not affect the motor function of mice.3.In formalin inflammatory pain model mice,CEL can inhibit the activation of CAMKII/ERK/CREB,and then down-regulate the expression of c-Fos,so as to increase the expression of strong enkephalin /κ-receptor and produce analgesic effect. |
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