Experimental Study of Yuxuebi Tablets by Inhibiting Peripheral Inflammation to Relieve Hyperalgesia and Foot Swelling in Mice with Chronic Inflammatory Pain / 中国实验方剂学杂志

Objective:To observe the effect of Yuxuebi tablets on hyperalgesia and foot swelling in mice with chronic inflammatory pain， and to explore the preliminary mechanism of action. Method:A mouse model of chronic inflammatory pain was established with left plantar injection of complete Freund's adjuvant （CFA）. The mice were divided into model group， positive drug ibuprofen group （91 mg·kg^-1）， Yuxuebi tablets low， medium and high dose groups （55， 110， 220 mg·kg^-1），with the sham operation group as the control. After successful modeling， the daily dose was divided into two doses in the morning and evening by gavage to give Yuxuebi tablets or ibuprofen to the stomach for a total of 19 days. On the 18^th day after the administration， the thermal pain threshold was detected by the hot plate method. On the 19^th day， the standard Von Frey fiber needle was used to detect the mechanical pain threshold of the mice， and the degree of foot swelling was scored and photographed. The liquid-phase suspension chip technology was used to quantitatively analyze 36 classic broad-spectrum inflammation-related factors like inflammatory factors and receptors. Bioinformatics were used to screen core targets and perform enzymelinked immunosorbent assay （ELISA） detection. Result:Compared with the sham operation group， the mechanical pain threshold and foot swelling score of the model froup significantly increased （<italic>P</italic><0.01）， the latent time of heat sensitivity significantly decreased（<italic>P</italic><0.01）， the expressions of 30 inflammatory factors in the foot increased（<italic>P</italic><0.05）. Compared with the model group， the high dose of Yuxuebi tablets significantly reduced the mechanical pain threshold and foot swelling score of mice with chronic inflammatory pain（<italic>P</italic><0.01）， significantly increased the latent time of heat sensitivity（<italic>P</italic><0.05）， and reduced the expressions of 30 inflammatory factors in the foot（<italic>P</italic><0.05）， among which tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-6 （IL-6）， interleukin-17A （IL-17A）， and C-C chemokine ligand 2 （CCL2） were the core targets screened out， and the expressions of TNF-<italic>α</italic>， IL-17A， and CCL2 significantly decreased （<italic>P</italic><0.01）. Conclusion:Yuxuebi tablets can relieve hyperalgesia and foot swelling in mice with chronic inflammatory pain， and its mechanism may be related to the inhibition of the expressions of peripheral inflammatory factors such as TNF-<italic>α</italic>， IL-17A， and CCL2 .

Relationship between complete Freund ' s adjuvant-induced chronic pain-induced negative emotions and morphological changes of hippocampal microglia / 解剖学报

Objective To investigate the alteration of mood and hippocampal microglia morphology in a mouse model of chronic inflammatory pain induced by complete Freund' s adjuvant (CFA). Methods Thirty-two male ICR mice were randomly divided into two groups, including normal saline control group (NS) and CFA model group (CFA). The pain model was established by right hindpaw intraplantar CFA injection. The change of mechanical pain threshold after CFA injection was measured by von Frey fiber needle, the locomotor activity and anxiety-like behavior were determined by open field test (OFT), the depression-like behavior was determined by sucrose preference test (SPT) and forced swimming test (FST). The expression of microglia marker ionized calcium binding adaptor molecule-1 (IBA-1) in the hippocampus was determined by immunohistochemistry and its morphological change was analyzed by Sholl analysis. Results Compared with the NS group, the mechanical pain threshold of CFA group decreased significantly (P<0.01). The behavior result showed that the CFA group showed remarkably reduced time in the inner area (P<0.01) compared with the NS group in the open field test; In the sucrose preference test, the percentage of sucrose preference (P<0.01) of CFA mice decreased significantly compared with the NS mice, while the immobility time of CFA mice (P<0.01) increased significantly in the forced swimming test compared with the NS mice. The immunohistochemistry showed that the number of microglia in the dentate gyrus (DG) of CFA mice increased significantly compared with the NS mice. The Sholl analysis result showed that compared with the NS mice, the number of intersections of microglia in hippocampal DG decreased significantly in CFA mice. Conclusion Our finding indicates that the negative emotions in CFA-induced chronic inflammatory pain may be related to the morphological changes of hippocampal microglia in the mice.

Analgesic effect of Fengshi Qutong Capsules on mice with chronic inflammatory pain caused by complete Freund's adjuvant / 中国中药杂志

The aim was to observe the analgesic effect of Fengshi Qutong Capsules(FSQTC) on chronic inflammatory pain in mice, and investigate its effect on p-ERK/COX-2 signal molecular activity. A model of chronic inflammatory pain was induced in mice by complete Freund's adjuvant(CFA). The mice were divided into normal control group, model group, model+FSQTC 0.3, 0.6 and 1.2 g·kg~(-1 )groups, model+positive control drug ibuprofen(IBP, 0.34 mg·kg~(-1)·d~(-1)) group, and normal control+ FSQTC 1.2 g·kg~(-1)group. FSQTC or IBP was given once a day by oral administration. Standard Von Frey fiber was used to evaluate the mechanical pain threshold, and the acetone stimulation was used to induce inflammatory plantar and observe the cold pain reaction scores. The mechanical pain threshold and cold pain reaction scores were observed before administration and 1, 2, 3, 4, 6 h after administration on the first day, as well as 3 h after administration on the 3 rd to 7 th day. The protein levels of PGE_2, COXs-1,2 and p-ERK in the spinal cord of the inflammatory foot and lumbar 4-5 were detected by enzyme-linked immunosorbent assay, Western blot, immunohistochemistry and immunofluorescence. The results showed that the mechanical pain threshold of the model group decreased and the cold pain reaction score increased as compared with the normal group. FSQTC application could dose-dependently increase the mechanical pain threshold and decrease the cold pain reaction score. The effect lasted for 6 h, most significant at 3 h. The effect of ibuprofen was similar to that of the 0.6 g·kg~(-1) dose group. In addition, FSQTC could reduce the abnormally increased protein content of PGE_2, COX-2 and p-ERK in the inflammatory foot and/or spinal cord of the model group, and the effect was most significant in middle and high dose groups. However, it had no effect on COX-1 in the inflammatory foot and spinal cord of mice. The results suggest that FSQTC has ob-vious analgesic effect on chronic inflammatory pain in mice, which may be related to inhibition of p-ERK/COX-2 signaling pathway.

Analgesic Effect of Panlongqi Tablet on Rats with Chronic Inflammatory Pain / 中国实验方剂学杂志

Objective:To observe the analgesic effect of Panlongqi tablet（PLQT） on rats with chronic inflammatory pain， and to explore mechanism of the action preliminarily from the perspective of the nuclear factor kappa-light-chain-enhancer of activated B cells （NF-κB）and mitogen-activated protein kinase（MAPKs） signaling pathways. Method:Rats were induced to establish model of chronic inflammatory pain by complete Freund adjuvant（CFA）， which was divided into normal group， model group， the PLQT 0.16，0.32，0.64 g·kg-1 group， and the ibuprofen 0.05 g·kg-1 group（also positive group）， give the medicine once a day by gavage. Standard Von Frey fiber was used to evaluated the mechanical pain threshold， acetone was used to stimulated rats inflammatory foot to get the cold-induced response score， with the mechanical pain threshold and cold-induced response score to be observed at 1， 2， 3， 4 and 6 h before and after administration on day 1， and at 4 h after administration on day 3-7. The content of PGE2， IL-1， TNF-α in serum， inflammatory foot and 4-5 lumbar spinal cord was detected by enzyme-linked immunosorbent assay（ELISA）. The protein level of MAPKs （p-p38， p-ERK， p-JNK） in lumbar spinal cord 4-5 was detected by Western blot. The expression of NF-κB p65 in the lumbar spinal cord was detected by IFA. Result:Model group had lower mechanical pain threshold and higher cold-induced response score than these in normal group（P<0.01）， while the mechanical pain threshold and cold-induce response score of the model rats were dose-dependent better regulated after administration of PLQT 0.16， 0.32， 0.64 g·kg-1·d-1（P<0.05，P<0.01）， these effect lasted 6 h， of which PLQT groups get the most significant effect on 4 h， however the effect of IBP was similar to that of PLQT medium dose group. In addition， PLQT reduced the abnormal increase of PGE2， IL-1 and TNF-α contents in serum， inflammatory foot and spinal cord of rats in model group， decreased the protein phosphorylation levels of ERK and JNK in spinal cord， and decreased the protein expression of NF-κB p65， that was significant in the PLQT high-dose group（P<0.01）. Conclusion:PLQT had significant analgesic effect on chronic inflammatory pain model rats， which may be related to the inhibition of NF-κB and MAPKs signaling pathways in spinal cord.

Effect of electroacupuncture on expression of GABA receptor mRNA in different brain regions in rats with chronic inflammatory pain / 中国针灸

OBJECTIVE@#To observe the expression of GABA receptor mRNA in different brain regions of the central nervous system in chronic inflammatory pain rats and the intervention effect of electroacupuncture (EA).@*METHODS@#A total of 48 SPF male SD rats were randomly divided into a blank control group, a model control group, an EA group and a sham EA group, 12 rats in each group. The model of chronic inflammatory pain was established by injecting Freund's complete adjuvant into the foot. The EA group was treated with EA 28 days after the model establishment. The "Housanli" (ST 36) and "Kunlun" (BL 60) were selected and treated with dilatational wave, 2 Hz/100 Hz in frequency, 0.5-1.5 mA for 30 min; EA was given only once. In the sham EA group, the same acupoints were selected but the needles were only inserted into subcutaneous area; EA was connected for 30 min without electrical stimulation. The behavior changes of mechanical pain threshold and thermal pain threshold before model establishment, 1 day, 3 days, 7 days, 14 days, 21 days and 28 days after the model establishment as well as emotional behavior 29 days after the model establishment were observed; the relative expressions of GABA receptor mRNA in anterior cingulate cortex, amygdala and hypothalamus were observed.@*RESULTS@#Compared with the blank control group, the change rates of mechanical pain threshold and thermal pain threshold in the model control group were decreased significantly 1 day, 3 days, 7 days, 14 days, 21 days, 28 days after model establishment (0.05). Compared with the blank control group, the expression of GABA receptor mRNA in the amygdala was decreased significantly in the model control group (<0.01); compared with the model control group and the sham EA group, the expression of GABA receptor mRNA in amygdala was increased after intervention in the EA group (<0.01).@*CONCLUSION@#Single treatment of EA could significantly increase the mechanical pain threshold and thermal pain threshold, improve abnormal emotional behavior in rats with chronic inflammatory pain, which may be related to the increasing of expression of GABA receptor mRNA in the amygdala.

Study on the Effects of 8-O-acetyl-shanzhiside Methylester on the Expression of HDAC 1-5 in the Spinal Dorsal Horn of Rats with Chronic Inflammatory Pain and Its Relationship with JAK 2-STAT3 Signaling Pathway / 中国药房

OBJECTIVE： To study the effects of 8-O-acetyl-shanzhiside methylester （8-OaS） on the expression of histone deacetylase 1-5 （HDAC1-5） in the spinal dorsal horn of chronic inflammatory pain model rats， and its relationship with Janus-activated kinase 2-signal transductions and activators of transcription 3 （JAK2-STAT3） signaling pathway. METHODS： SD rats were randomly divided into normal control group， sham operation group （normal saline）， complete Freund’s adjuvant （CFA） group （normal saline）， 8-OaS low-dose， medium-dose and high-dose groups （2， 20， 200 μg/kg）， with 6 rats in each group. Except for normal control group and sham operation group， chronic inflammatory pain model was induced by subcutaneous injection of CFA into the left hind toe of rats in other groups； after modeling， those groups were given relevant medicine intraperitoneally， once a day， for consecutive 7 d. Thermal radiation method was used to detect the latency of paw withdraw in rats on the 1st， 2nd， 3rd， 4th， 5th， 6th， 7th， 8th， 11th and 15th day of administration. Rats were grouped and given medicine according to the above method of the latter 5 groups. The protein expression of HDAC 1-5， phosphorylated JAK2 （pJAK2） and phosphorylated STAT3 （pSTAT3） in the spinal dorsal horn of lumbar enlargement segment in rats were detected by Western blot method after last medication. Rats were randomly divided into sham operation group （normal saline）， CFA group （normal saline）， 8-OaS group （20 μg/kg） and JAK2-STAT3 inhibtor AG490 group （8 mg/kg）， with 6 rats in each group； IP model was established by same method as above and then were given relevant medicine intraperitoneally， once a day， for consecutive 7 d. The expression of HDAC5 and glial fibrillary acidic protein （GFAP） in spinal dorsal horn of rats were detected by immunofluorescence histochemical staining. RESULTS： Compared with normal control group and sham operation group， the latency of paw withdraw was shortened significantly in other groups （P＜0.05）. Compared with CFA group， the latency of paw withdraw was prolonged significantly in 8-OaS low-dose， medium-dose and high-dose groups （P＜0.05）， in dose-dependent manner. Compared with sham operation group， the protein expression of HDAC 1-5， pJAK2 and pSTAT3 in spinal dorsal horn of rats were increased significantly in CFA group （P＜0.05）. Compared with CFA group， the protein expression of HDAC5， pJAK2 and pSTAT3 in spinal dorsal horn of rats were decreased significantly in 8-OaS low-dose， medium-dose and high-dose groups （P＜0.05）， but there was no statistical significance in the protein expression of HDAC 1-4 （P＞0.05）. HDAC5 was expressed on astrocytes in the spinal dorsal horn； compared with sham operation group， the expression of GFAP and HDAC 5 were increased significantly in spinal dorsal horn of rats in CFA group （P＜0.05）. Compared with CFA group， the expression of GFAP and HDAC5 in spinal dorsal horn of rats were decreased significantly in 8-OaS group and AG490 group （P＜0.05）. CONCLUSIONS： 8-OaS can effectively relieve CFA-induced chronic inflammatory pain， the mechanism of which may be associated with the down-regulation of HDAC5 expression and the phosphorylation levels of JAK2 and STAT3.

The effects of anthocyanin on chronic inflammatory pain induced by complete Freund's adjuvant and its mechanism / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects and mechanisms of anthocyanin from Ligustrum vicaryi on chronic inflammatory pain induced by complete Freund's adjuvant.@*METHODS@#Thirty male SD rats were randomly divided into three groups (=10):normal saline control group (NS), chronic inflammatory pain model group(Mod, injected with complete Freund's adjuvant(CFA) 100 μl to the left hind leg), anthocyanin treatment group(Ant, dosed with anthocyanins (90 m), mechanical pain threshold (MPT), and left toe volume in each group were measured before modeling and 1,3,5,7,9,11,13 days after operation. Antioxidant indexes in serum were mensurated by spectrophotometer, and the total capsaicin receptor (TRPV1) and phosphorylated capsaicin receptor (p-TRPV1) in hippocampus were detected by Western blot.@*RESULTS@#In comparison with controls, HPT and MPT were improved (<0.05),toe swelling was reduced(<0.05), the serum level of SOD was increased (<0.01), while the levels of MDA and NO were decreased (<0.05), the ratio of P-TRPV1/TRPV1 protein was depressed in Mod rat hippocampal region treated with anthocyanin.@*CONCLUSIONS@#The results show that anthocyanins has an analgesic effect on chronic inflammatory pain induced by CFA, and its mechanism may be related to the improvement of antioxidant capacity and the reduction of TRPV1 phosphorylation.

Tat-LK15-mediated delivery of nNOS siRNA in rats with chronic inflammatory pain / 实用医学杂志

Objective To investigate the potential application of a non-viral gene carrier , TAT-LK15 , for delivering nNOSsiRNAin vivo and to study whether TAT-LK15/siRNA can be a new treatment method for chronic inflammatory pain. Method TAT-LK15 was complexed with nNOSsiRNA or scrambled control siRNA. The expression of nNOS was determined in SCDH of chronic inflammatory pain rats by western-blot assay. Pain control efficacy was evaluated by mechanical withdrawal threshold (MWT) and thermal withdrawal duration (TWD) assays. Results nNOS protein expression was efficiently inhibited by intrathecal injection of TAT-LK15/siRNA complexes , with the reduction of nNOS protein by 52%. Moreover , injection of TAT-LK15/siRNA com-plexes significantly could decrease MWT , but increase TWD in rats with chronic inflammatory pain. Conclusions TAT-LK15 can efficiently deliver nNOSsiRNAin vivo and nNOSsiRNA can relieve chronic inflammatory pain in rats.

SiRNA interference on expression of MrgC and phosphorylation of PKCεin ipsilateral dorsal root ganglion of rats with chronic inflammatory pain / 中国比较医学杂志

Objective To observe the Interference effects of siRNA( small interference RNA) intrathecal injection on the expression of mRNA and protein of MrgC, on PWTs ( paw withdrawal thresholds) and the phosphorylation of PKCεSer729 in ipsilateral DRG( dorsal root ganglion) of rats with chronic inflammatory pain induced by CFA( complete freund’ s adjuvant).Methods 16 health adult male SD (Sprague-Dawley) rats were randomly divided into 2 groups: control siRNA group and MrgC siRNA group, 8 rats in each group.After success of intrathecal catheterization, corresponding siRNA was injected in rats for 4d, once a day, 5μg/d per rat.The model of chronic inflammatory pain was established by CFA (0.1ml per rat) subcutaneously injected into the right hind paw at 4th day post-administration, then two groups were administrated corresponding siRNA on alternate day and executed at the 11th day post-administration.The PWTs were measured at 5 time points of pre-intrathecal catheterization, pre-administration, 4th day post-administration(0h post-CFA injection), 5th day post-administration(24h post-CFA injection), 11th day post-administration(7 d post-CFA injection). The expression of MrgC mRNA in ipsilateral DRG was detected by fluorogenic quantitative PCR, and the expression of MrgC and the phosphorylation of PKCε Ser729 in ipsilateral DRG was detected by immumofluorescence method.Result Compared with 4th day post-administration, PWTs of both two groups at 5th day post-administration decreased significantly ( P<0.01 ) .While there was no significant difference of PWTs between two groups at every detective time point. Compared with control siRNA group, the expression of MrgC mRNA and the rate of MrgC positive cells in MrgC siRNA group both decreased significantly ( P<0.01,P <0.05), whereas the rate of p-PKCε Ser729 positive cells increased obviously ( P<0.05) at 11th day post-administration.Conclusion MrgC siRNA can effectively interfere the expression of mRNA and protein of MrgC in L4-L6 ipsilateral DRGs of rats with chronic inflammatory pain induced by CFA, and the siRNA interference on MrgC can obviously up-regulate the phosphorylation of PKCε Ser729, while it has no significant effect on PWTs of rats.