Chronotherapeutics Study Of SIN And T-614 In Cillagen Induced Arthritis

Objectives Sinomenine(SIN)or Iguratimod(T-614)were used at different times to analyze the day and night differences in the treatment of collagen-induced arthritis(CIA)and related toxic and side effects,and its possible mechanism provides experimental basis for further clinical application of time therapeutics.Methods 1 Study on time therapy and its mechanism of sinomenine and elamud in CIA rats: 80 healthy SPF-grade male Wistar rats weighing 120±10g were housed in the Time Biology Laboratory [from 08:00~20:00 during the Ming Period,20:00~08:00 from the Dark Period,or from the Zeitgeber Time,ZT),ZT0~ZT12 was selected as the experimental period.free drinking water,and adapted for feeding for 10 days.Then,80 rats were randomly divided into 70 model groups and 10 blank(NG groups).Rats in the model group were subcutaneously injected with bovine type II collagen(CII)on d0 and 14 d,respectively,under sterile conditions to establish a CIA model.The right hind paw volume and body weight were measured every 7 days during the experimental period(0d-d50).Rats with CIA model were successfully established on 21 d,and were divided equally into SIN-ZT0,SIN-ZT12,T-614-ZT0,T-614-ZT12 and model group(M)according to the arthritis score.Each group had 10 rats.Group 10 only.Drug intervention was started at d22,sinomenine was given intragastricly to sinomenine(150 mg/Kg·d-1,2 m L),and T-614 treatment group was given gavage with irammodole(6 mg/Kg·d-1);2 m L),M group and NG group were intragastrically administered with an equal volume of sodium carboxymethyl cellulose for 28 days.D49 Photographs of rat paw X-rays,d50 in ZT0-ZT3.Rats were sacrificed by abdominal anaesthesia with 2% pentobarbital.Blood was taken,serum was separated,cryopreserved and prepared for examination;ankle joints were taken and fixed.Decalcified,paraffin-embedded,paraffin tissue sectioned for use.Serum levels of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),and melatonin(MLT)were determined by enzyme linked immunosorbent assay(ELISA).Corticosterone(CORT)content,hematoxylin-eosin staining(HE staining)micrographs were used to observe the pathological changes of foot and ankle synovial membrane,and immunohistochemical technique(SP technique)was used to detect TNF-? and IL-1? in the athlete's foot.Osteoprotegerin(OPG),Receptor activator for nuclear factor-?B ligand(RANKL),Clock gene expression protein(CLOCK,BMAL1)expression.2 A preliminary study on the time toxicology of sinomenine and iguratimod: Sixty healthy SPF-grade male Kunming mice(body weight 20±10g)were housed in the above time biology laboratory.Adapted to feed for 10 days,free access to drinking water.After the mice were randomly assigned to SINZT0,SIN-ZT12,T-614-ZT0,T-614-ZT12,NG group,12 in each group.d0~d21 for drug intervention,SIN time toxicology group was given intragastric administration of sinomenine(150mg/Kg·d-1;0.9m L),T-614 time toxicology group was administered intragastrically to iamomod(6mg/ Kg·d-1;0.9 m L),NG group was intragastrically administered with equal volume of sodium carboxymethyl cellulose.d22 The mice were sacrificed by ZT0-ZT1 decapitation,blood was collected,plasma was separated and cryopreserved.Alanine transaminase(ALT)and acute erythrocyte transaminase(AST)levels in T-614-ZT0,T-614-ZT12 and NG groups were detected by microplate method.SIN-ZT0 and SIN-were measured by ELISA.Plasma Histamine(HIS)levels in ZT12 and NG groups.All data were processed using IBM SPSS21.0 software,and the measurement data was in accordance with the normal distribution data expressed as mean±standard deviation((?)±s).One-way analysis of variance was used for comparison among multiple groups.Comparison was made between the two groups.LSD was used to test the variance at the same time.Dunnett's T3 method was used to test the variance,and the test standard was ?=0.05.Results 1 Study on time therapy and its mechanism of sinomenine and elamud in CIA rats: 1.1 General growth status and weight comparison: Prior to administration,arthritic rats were more irritated,had poorer hair luster,hair loss,and apathetic,and the fight within the group was more frequent.Some of the rats had limited mobility due to swelling or pain in the paws,or joint stiffness.The pain or chronic inflammation resulted in less water and thinner body.After treatment in each treatment group,dietary water was more frequent than before administration,hair loss was reduced,and gloss was increased.The activity of rats gradually increased,the fighting phenomenon in the group decreased,and the joint stiffness improved.The body weight gain of rats in group M was delayed after two CII immunizations.After drug intervention,the weight gain of rats in the SIN-treated group was slower than that of the NG group.On days DAY42 and DAY49,the weight of rats in the SIN-treated group was higher than that in the M group;The body weight of SIN-ZT0 rats was always lower than that of SIN-ZT12 group,and the weight difference between the two groups gradually became larger.The weight gain of T-614 treatment group was slower than that of NG group.The body weight of M group rats was lower than that of T-614 treatment group at the beginning of DAY35.The body weight of T-614-ZT12 group was significantly higher than that of M group at DAY42 and DAY49.P<0.05);DAY42,T-614-ZT8 group rats significantly higher body weight than M group(P <0.05);but T-614-ZT0 group rats began at DAY35,body weight is always lower than T-614-ZT12 group.1.2 Change in right foot volume in the time treatment group: After SIN intervention,the paw volume of rats decreased rapidly,and the paw volume of rats in SIN-ZT0 and SIN-ZT12 groups at DAY35 and DAY42 was significantly lower than that of M group(P<0.05);at DAY35,DAY42,and DAY49,SIN-ZT12 group's paw volume was always lower than the SIN-ZT0 group.After T-614 intervention,the paw volume of rats rapidly decreased.On DAY28~DAY42,the paw volume in M group was significantly higher than that in T-614-ZT0 and T-614-ZT12 groups(P<0.05).DAY49,M group,foot The paw volume was significantly higher in the T-614-ZT12 group(P<0.05),and in the DAY35-DAY49 group,the paw volume in the T-614-ZT12 group was always lower than that in the T-614-ZT0 group.1.3 Joint Imaging Changes: After 4 weeks of administration,the iliac articular surface was obscure in the M group.The bone defect was obvious,and the soft tissue swelled more obviously.The gap between the metacarpophalangeal joints of the hind paw was evenly narrowed and the osteoporosis was obvious.Most of the rat's front paws also showed different degrees of articular bone.Mass destruction,narrowing of gaps,soft tissue swelling,etc.In the SIN-ZT0 group,the sacroiliac joint destruction and articular surface blur degree were light,some bone defects were seen in the joints,joint space was narrowed,and osteoporosis was found.The sacroiliac joint surface of the SIN-ZT12 group was still visible,and the degree of bone destruction was lighter than that of SIN-ZT0 group.In the T-614-ZT0 group,the sacroiliac joint surface was visible,and the sacroiliac joint space was narrowed with osteoporosis;the T-614-ZT12 group was visible on the sacroiliac joint surface and the degree of bone destruction was higher than that of the T-614-ZT0 group.Light.1.4 Rat serum TNF-?,IL-1?,melatonin and corticosterone content: After 4 weeks,the serum levels of cytokines(TNF-?,IL-1?)and melatonin(MLT)in group M were significantly higher than those in NG group(P<0.01),and the content of serum corticosterone(CORT)was higher than that of NG group.Significantly decreased(P<0.01);serum levels of cytokines(TNF-?,IL-1?)and melatonin in the SIN and T-614 treatment groups were significantly lower than those in the M group(P<0.01,P<0.05).Serum corticosterone levels increased significantly(P<0.01).After 4 weeks of intervention with SIN and T-614,the serum levels of cytokines(TNF-?,IL-1?)were significantly lower in the ZT12 group than in the ZT0 group of the SIN and T-614(P<0.05);each of the SIN and T-614 The serum melatonin level in ZT12 group was higher than that in SIN and T-614(P<0.05),but there was no significant difference in SIN and T-614 between ZT12 group and NG group(P>0.05);SIN and T-The levels of serum corticosterone in each ZT12 group were higher than those in ZT0 group of SIN and T-614(P<0.05).1.5 Ankle joint pathological changes: In M group,4 to 7 layers of synovial cells with loosely arranged and disordered synovial cells were observed by HE staining of synovium of the ankle joint.Synovial cell papillary hyperplasia,lymphocyte infiltration,and lymphocyte aggregation were partially observed.In severe cases,multinuclear macrophage cells could be seen.Synovial vascular hyperplasia,vasospasm formation erosion of trabecular bone,cartilage bone destruction.In the SIN and T-614 treatment groups,the synovial hyperplasia of the ZT12-administered group was lighter than that of the ZT0-administered group,and the synovial cell lymphocyte infiltration was lighter;synovial cell papillary hyperplasia was even seen,but still administered as ZT0.The group is heavy.1.6 Expression of TNF-?,IL-1?,OPG,RANKL,CLOCK and BMAL1 in Ankle Joints: The expression of TNF-?,IL-1?,OPG,RANKL,CLOCK and BMAL1 in the ankle joint of group M was significantly higher than that of NG group,and the expression of OPG was lower than that of NG group;TNF-? was compared between SIN and T-614 treatment group and M group.The expression of IL-1?,RANKL,CLOCK and BMAL1 was significantly decreased,and the expression of OPG was higher than that of M group.In the SIN and T-614 treatment groups,except for OPG,ZT0 group had higher expression than ZT12 group,and OPG expression had ZT12.The group is higher than the ZT0 group.2 A preliminary study on the time toxicology of sinomenine and iguratimod: 2.1 General growth and weight changes in mice: The body weight of mice in NG group increased rapidly in the first week,and the growth rate in the second and third weeks gradually slowed down and was more active.The mice in the SIN and T-614 toxicology groups had a slower growth than the NG group,with the ZT12 group having the slowest growth rate;the first and second week activities were more frequent after the intervention,and the activity was less after the intervention than the NG group;the T-614 toxicology group Fighting phenomenon within the group and scratches on both sides of the upper limb lead to hair loss.After SIN intervention,the body weight of SIN14 and DAY21 groups was significantly lower than that of the blank group(P<0.05).The body weights of DAY7,DAY14,and T-614-ZT12 groups after T-614 intervention were significantly lower than those of NG group(P<0.01).In the SIN and T-614 toxicology groups,the weight of the ZT12 group was always lower than that of the ZT0 group.2.2 Plasma histamine content after sinomenine intervention: After 3 weeks of SIN intervention,plasma histamine content in SIN-ZT0 and SIN-ZT12 groups showed higher levels,which were significantly higher than those in NG group(P<0.05,P<0.01).Among them,SIN-ZT12 group was significantly higher than SIN-ZT0 group(P<0.05).2.3 The plasma AST and ALT levels in mice after the intervention of iguratimod: Three weeks after T-614 intervention,plasma AST levels were higher in the two groups than in the NG group.The T-614-ZT12 group was significantly higher than the NG group(P<0.05);the T-614-ZT12 group was higher than the T-614-The ZT0 group is slightly higher.Plasma ALT levels in both groups were significantly higher than those in the NG group(P<0.01).Plasma ALT levels were slightly higher in the T-614-ZT12 group than in the T-614-ZT0 group.Conclusions 1 The sinomenine and ilamomod intervened in CIA rats at different times during the day and night.The therapeutic effect was different between day and night.The reasons were as follows: 8 points earlier to give drug intervention 8 serum and arthritis cytokines(TNF-?,IL-1?)low expression and high corticosterone expression,synovial OPG high expression,RANKL low expression.Its mechanism may be related to the melatonin,clock gene regulation of diurnal and corticosterone secretion or regulation of inflammatory cytokine expression levels.2 Sinomenine and irammod interfered with normal Kunming mice at different times during the day and night.The expression of histamine in the sinomenine late 8 group was higher than that in the early 8 o'clock group;the damage of liver function was found in the 8th group of irammod.AST,ALT)is slightly higher than the early 8-point group,and its mechanism needs further study.