| Objective:To observe the expression of NLRP3 inflammasome and pain-causing factors in KOA guinea pigs with kidney deficiency,to explore the corresponding changes after the intervention of Bushen Yiqi Recipe and MCC950,and then to investigate the effect of Bushen Yiqi Recipe-containing serum and MCC950 on the expression of NLRP3 inflammasome and pain-causing factors of macrophages and fibroblast-like cells.MethodsFirstly,the KOA guinea pig model with kidney deficiency type was established to evaluate its changes of synovial inflammation,pain and NLRP3 inflammasome,and then the Bushen Yiqi recipe and NLRP3 inflammasome-specific inhibitor MCC950 were intervened it to evaluate the changes of the synovial inflammation,pain,and NLRP3 inflammasome.Finally,the changes of NLRP3 inflammasome and pain factors were evaluated after the intervention of Mac and FLS with Bushen Yiqi recipe-containing serum.The specific methods are as follows:1.We established and evaluated the KOA guinea pigs model of kidney deficiency and observed the expression of NLRP3 inflammasome and pain factors.The 4-month-old ordinary Hartley female guinea pigs were randomly divided into a sham operation group(Sham)and a castration group(OVX),and the kidney deficiency model was established by removing both ovaries.The kidney deficiency model was evaluated by observing the general condition of guinea pigs,body weight changes and the detection of serum E2;the knee joint gross view,pathological section staining and semi-quantitative scoring of cartilage and synovium,and micro-CT micro-quantitative changes in cartilage and subchondral bone were evaluated for the early-mid KOA model and synovitis;Synovial inflammatory pain was assessed by static bipedal balance or dynamic plantar Von Frey fiber,IL-1β and NGF of serum,and WB and RT-PCR detection of NLRP3 inflammasomes in spinal cord,dorsal spinal root nerves(DRGs)and synovial membrane.2.To investigate the effect of Bushen Yiqi Recipe on NLRP3 inflammasome in KOA guinea pigs with kidney deficiency.The 4-month-old ordinary guinea pigs were randomly divided into five groups:sham operation group(Sham,J)、model group(model,M),regular dose of Bushen Yiqi recipe(C)group,high dose of Bushen Yiqi recipe(G)group and diacerein(Y)group,which were given continuous gavage for 30 days after modeling.Serum,synovium,cartilage,spinal cord and spinal dorsal root nerve were collected,and the detection methods were the same as before,and the changes of NLRP3 inflammasome and pain factors in guinea pigs after Bushen Yiqi Recipe were observed.Changes in renal insufficiency after treatment were assessed by general behavioral observations and body weight changes.Post-treatment cartilage and subchondral bone changes were assessed by histopathology and imaging.3.We explored the effect of MCC950 inhibition of NLRP3 inflammasome on pathological changes in KOA guinea pigs.The 4-month-old guinea pigs were randomly divided into five groups:sham operation group(Sham,J),model group(model,M),normal dose of Bushen Yiqi recipe+normal saline(CW)group,conventional dose of Bushen Yiqi recipe+MCC950(CM)group and MCC950(MCC)group.Five days before the sampling,the CM group and MCC950 group were given a certain amount of intraperitoneal injection of MCC950,once a day,for 5 consecutive days,and the CW group was given the same amount of normal saline intraperitoneal injection.The methods of detection and evaluation were the same as before.4.To investigate the effect of Bushen Yiqi Recipe containing serum and MCC950 on NLRP3 inflammasome in Mac and FLS.Primary Mac and FLS were extracted from 4-month-old female guinea pigs in batches.Macs after LPS intervention were divided into six groups for drug-containing serum and MCC950(MCC-H)intervention,respectively.The five groups of medicated serum were:sham operation group(Sham,J),model group(M),normal dose of Bushen Yiqi prescription+normal saline(CW)group,normal dose of Bushen Yiqi prescription+MCC950(CM)group and MCC950(MCC)group.The activated Mac cell supernatants were intervened in FLS,and the intervention groups were the same as Mac.CCK8 detection was used to explore the appropriate concentration of drug-containing serum and MCC950 dosage;WB detection of NLRP3 inflammasome was used to explore the intervention time.The elisa of Mac supernatant and WB and RT-PCR assay of NLRP3 inflammasome were used to evaluate the changes of cellular NLRP3 inflammasome after intervention.Results1.The guinea pigs in the OVX group showed obvious signs of kidney deficiency such as hair loss,arched back,loose stools and poor diet.The weight gain and growth rate were significantly reduced,and the serum E2 was significantly reduced,indicating that the kidney deficiency model was successful.The bipedal balance test found that there was no significant difference in the weight-bearing of the left and right lower limbs of the guinea pigs.The Von Frey fiber plantar pain measurement results showed that compared with the Sham group,the PWT value of the OVX group was significantly reduced from the first week after the operation.The histological results of the knee joint showed that compared with the Sham group,the subpatellar synoviums in the OVX group increased,the synovial fluid was more,the cartilage surface was incomplete,cracked or missing,and the local area was obviously thinned.Under the light microscope,the cartilage surface of the sham group showed punctate loss,the tide line was visible but not clear enough,and the surface matrix was partially dissolved;but in the OVX group,the cartilage surface was missing,the tide line was obviously pale and incomplete,the cartilage structure was partially missing,and some cells and matrix were significantly lysed,and the cells were of different shapes and sizes and irregularly arranged.Further semi-quantitative scoring showed that compared with the Sham group,the cartilage gross view,synovitis and Krenn scores in the OVX group were significantly increased,and the scores were statistically significant.The P-value for the difference was 0.051.These evidences support the establishment of the KOA model.Serological Elisa test found that:IL-1β and NGF were significantly increased in OVX group.The WB detection results of spinal cord,GRDs and synovial tissue showed that compared with Sham group,the protein expressions of caspase1-p20 in spinal cord,GRDs and synovial tissue of OVX group were significantly up-regulated,and that of NLRP3 and IL-1 β were significantly up-regulated in synovial tissue and GRDs of OVX group.But there was no statistical difference in the expression of IL-1β spliceosome.The expression of NGF was significantly up-regulated,but there was no difference in expression of NLRP3 and IL-1β precursor in spinal cord.But IL-1β spliceosomes in spinal cord and DRGs were not released.The RT-PCR results of spinal cord,GRDs and synovial tissue showed that compared with the Sham group,the expressions of NLRP3,caspase1,IL-1β and CCL17 in the spinal cord,GRDs and synovial tissue of the OVX group were significantly up-regulated.The expression of NGF was up-regulated but not statistically different.Overall,the protein and gene levels of NLRP3,caspase1 and IL-1β in the synovium and GRDs of the OVX group were significantly up-regulated,and the NGF and IL-1βspliceosome were also up-regulated,but the differences were not statistically significant;the protein levels of NGF in the spinal cord,GRDs and synovial tissue of the OVX group were significantly up-regulated but the mRNA expressions were not different.The protein and gene levels of caspase1 in the spinal cord of the OVX group were significantly up-regulated,and the mRNA expressions of NLRP3 and IL-1 β were significantly different,but the protein levels were not.The results of micro-CT cartilage morphological analysis of the two groups showed that compared with the Sham group,the cartilage volume and thickness in the medial platform ROI area of the OVX group were significantly reduced,while the average surface areas were reduced but not statistically significant,indicating that the degradation of OVX guinea pig cartilage caused that the distribution of cartilage were differences and the total amount were significantly lost.Subchondral bone analysis showed that compared with the Sham group,the bone volume fraction,bone surface density,trabecular thickness and number of the ROI area of the medial platform in the OVX group decreased,and the SMI indexes increased,and the differences were significant;while BMD and BS/BV were no statistical difference.This indicated that the subchondral bone of KOA in the OVX group were in an active period of reconstruction and were dominated by bone resorption,which were also in line with the performance of KOA in the early and middle stages.2.After 2 weeks of gavage,the C and G groups occasionally had arched back or loose stools,rarely hair loss or poor diet,mainly manifested as clean body,shiny coat color,brisk movements,good diet,ruddy tongue and claws and significant weight gain and so on,while M group had more common hair loss or loose stools,and had a general diet and less weight gain.The results of Von Frey fiber pain measurement showed that compared with group M,the PWT values of groups C,G and Y increased significantly at the beginning of T2,and gradually increased with time,and the differences were statistically significant at T2 and T5,and the differences of group C and Y were statistically significant at T3.At T4,the PWT values of group Y increased significantly and were statistically significant.Serum ELISA test results showed that compared with the M group,the serum levels of IL-1β and NGF in the C,G and Y groups were decreased with statistical significance.The pathological results showed that compared with the M group,the modified Mankin and Krenn scores of the C,G and Y groups were significantly decreased but not statistically significant,while the OARSI-synovitis scores were significantly decreased with statistically significant.The WB results of synovial tissue showed that compared with the M group,the expressions of NLRP3,caspase1-p20 and IL-1β precursor protein were significantly decreased in groups C,G and Y,and the levels of NGF protein were significantly decreased in groups C and G with statistical significance;compared with group Y,the levels of NLRP3 were significantly up-regulated in group C,while the levels of NGF protein were significantly decreased in group G and C.RT-PCR test results showed:spinal cord tissue:compared with group M,the levels of caspase1 gene were significantly down-regulated in groups C and G,the levels of IL-1β gene were significantly down-regulated in groups C,G and Y,and the levels of CCL17 gene were significantly down-regulated in groups C and Y with statistical significance.The levels of NLRP3 and NGF gene were significantly down-regulated but not statistically different among the groups.GRDs:compared with group M,the levels of caspase1 in group G were significantly down-regulated,and the levels of NGF in groups C and G were significantly down-regulated with statistical difference,while the levels of NLRP3,IL-1β and CCL17 in each intervention group were significantly down-regulated with no statistical significance.Synovial membrane:compared with the M group,the levels of NLRP3 gene were significantly down-regulated in C,G and Y groups,the levels of caspase1 were significantly down-regulated in G and Y groups,the levels of NGF were significantly down-regulated with statistical significance in C and Y groups,while the levels of IL-1β and CCL17 were significantly down-regulated with no statistical significance.Overall,compared with the M group,the protein expressions of NLRP3,caspase1-p20,IL-1β precursor and NGF in synovium were significantly down-regulated in the C group,and the gene expressions of NLRP3 and NGF in synovium were significantly down-regulated in the C group;compared with the M group,spinal caspase1 and IL-1β genes in group C were significantly down-regulated,and the gene expressions of caspase1 and NGF in GRDs in group C were significantly down-regulated and statistically different;except that compared with group Y,in group C the protein expressions of NLRP3 were significantly up-regulated and NGF proteins were significantly decreased,the groups G and Y did not show a significant advantage.The results of micro-CT medial platform cartilage ROI analysis in each group showed that compared with the M group,the cartilage volumes in the C and G groups were significantly increased with statistical significance,the cartilage thicknesses in the C and G groups were significantly increased but no statistical significance.Compared with the Y group,the TV values of C and G groups were increased significantly but not statistically significant.The trend shows that Bushen Yiqi recipe could delay the degeneration of KOA cartilage,while diacerein has little effect on delaying the wear of KOA cartilage.The results of micro-CT subchondral bone ROI analysis in each group showed that compared with group M,BV/TV in group Y was significantly increased,and BS/TV and Tb.N in groups C,G and Y were significantly increased with statistical significance.It indicated that the C,G and Y groups could delay the process of KOA subchondral bone resorption to a certain extent.3.The results of Von Frey fiber pain measurement showed that,compared with the M group,the PWT of the CW,CM and MCC groups increased significantly at T2,the PWT of the CM and MCC groups increased significantly at T3,and the PWT of the CW,CM and MCC groups were significantly increased at T4 and T5.The ELISA detection of guinea pig serum in each group showed that compared with the M group,the IL-1β and NGF in the CW,CM and MCC groups were significantly decreased.It is puzzling that for NGF,both the CW and MCC groups were significantly and statistically significantly lower compared to the CM group.The histological scores of guinea pig cartilage in each group showed that compared with the M group,the cartilage and synovial scores of the CW,CM and MCC groups were significantly decreased with no statistical significance,which may be related to individual differences and insufficient samples.The results of synovial tissue WB were as follows:compared with the M group,the protein expressions of NLRP3,caspase1-p20,IL-1β precursor and NGF in the CW,CM and MCC groups were significantly decreased.The expressions of IL-1 β precursor were significantly decreased in the MCC group,and the expressions of NLRP3 were significantly decreased in the MCC group.Compared with the CM group,the IL-1β precursors were significantly increased in the MCC group.RT-PCR detection results showed:spinal cord tissue:compared with the M group,the caspase1 genes were significantly down-regulated in the CW and CM groups,the IL-1β genes were significantly down-regulated in the CW,CM and MCC groups,and the NLRP3,CCL17 and NGF genes were significantly down-regulated in each intervention group,but there were no statistical significance.GRDs:compared with the M group,the NLRP3,caspase1,IL-1βand NGF genes were significantly down-regulated in the CW,CM and MCC groups,and the CCL17 genes were significantly down-regulated in each intervention group with no statistical significance.Synovial tissue:compared with M group,the genes of NLRP3 and caspase1 were significantly down-regulated in CW,CM and MCC groups,and IL-1β,CCL17 and NGF genes were significantly down-regulated in each intervention group with no statistical significance.Overall,compared with the M group,the proteins and genes of NLRP3 and caspase1 in the synovium of the CM group were significantly down-regulated,and the protein levels of IL-1β and NGF were down-regulated,but there were no significant difference in the gene levels;The expressions of caspase1 and IL-1β in the spinal cord of the CM group were significantly down-regulated,and the gene expressions of NLRP3,caspase1,IL-1β and NGF in the GRDs of the CM group were significantly down-regulated,while CCL17 genes were significantly down-regulated with no statistical difference.The analysis results of cartilage ROI morphology in each group showed that compared with the CW group,the TVs of the M,CM and MCC groups were significantly reduced,and the thicknesses of the M and MCC groups were significantly reduced,and there were no significant difference between the other intervention groups.The results of the subchondral bone ROI morphology in each group showed that compared with the M group,the BV/TV of the CM and MCC groups were significantly increased,the BV/TV of the CM and MCC groups were significantly increased,and the BS/TV of the CW,CM and MCC groups were significantly increased.and Tb.N were significantly increased;compared with the CW group,the BV/TV in the MCC group were significantly increased,the BS/TV and Tb.N in the CM and MCC groups were significantly increased,and the Tb.Th in the CM group were significantly increased;There were no significant differences in BS/BV,SMI and BMD groups.This indicated that MCC950 treatment could delay the progression of KOA subchondral bone disease to a certain extent.4.ELISA showed that the levels of IL-1β and NGF in the supernatant of Mac cells were significantly different,especially compared with the M group,the CM,MCC and MCC-H groups were significantly lower,but the cell supernatant of FLS was not detected.The WB results of Mac showed that:compared with the M group,the expressions of NLRP3,caspase1-p20 and IL-1β precursor protein were significantly down-regulated in the J,CW,CM,MCC and MCC-H groups;compared with the CW group,in the J and MCC-H groups caspase1-p20 proteins were significantly down-regulated,caspase1-p20 proteins were significantly up-regulated in CM group,IL-1β precursor proteins were significantly down-regulated in J and MCC groups,and IL-1β precursor proteins were significantly up-regulated in CM group;compared with CM group,in the J,MCC and MCC-H groups caspase1-p20 and IL-1β precursor proteins were significantly down-regulated,and IL-1βprecursor protein expressions were significantly up-regulated in MCC-H group compared with MCC group.The RT-PCR results of Mac showed that compared with the M group,the expressions of NLRP3 gene were significantly down-regulated in the J,CW,CM,MCC and MCC-H groups,and the expressions of the NGF gene were significantly down-regulated in the J,CW,CM and MCC-H groups.Compared with the CM group,the expression of caspase1 genes were significantly down-regulated in MCC-H group,and there were no significant difference in the expressions of IL-1β and CCL17 among the groups.The WB results of FLS showed that compared with the M group,the expressions of NLRP3,caspase1-p20 and IL-1β precursor proteins in the J,CW,CM,MCC and MCC-H groups were significantly down-regulated with no statistical significance;The expressions of NLRP3 and IL-1β precursor protein were significantly down-regulated in MCC-H group,with statistical significance.The RT-PCR results of FLS showed that compared with the M group,the expressions of caspase1 in the J,CW,CM,MCC and MCC-H groups were significantly down-regulated,and the expressions of CCL17 in the MCC group were significantly down-regulated;compared with the CM group,the expressions of NGF in the MCC-H group were significantly increased.Compared with the MCC group,the NLRP3 gene expression was significantly up-regulated in the CW and MCC-H groups,and the caspase1 gene expressions in the CM group were significantly up-regulated.There were no significant difference between the IL-1β groups.Overall,for Mac and FLS,compared with the M group,the NLRP3 inflammasome protein and gene levels were significantly down-regulated among the groups,but there were differences in local comparisons,which needed further experiments or multiple repetitions to verify.Conclusion1.The protein and gene levels of NLRP3,caspase1 and IL-1 β were highly expressed in the synovium of KOA guinea pigs with kidney deficiency type,the protein and gene levels of caspase1 and IL-1β in GRDs were highly expressed,and the proteins of caspase1 and NGF in the spinal cord were highly expressed.The NLRP3 and NGF protein levels were significantly up-regulated in GRDs,but the mRNA expressions were not different,and the mRNA expressions of NLRP3 and IL-1β in the spinal cord were significantly different,but the protein levels were not different.2.Bushen Yiqi Recipe can reduce synovitis and pain response in KOA guinea pigs with kidney deficiency by regulating the protein and gene levels of NLRP3 inflammasome in the synovium,GRDs and spinal cord.3.Specific blocking of NLRP3 and its inflammasome by MCC950 can significantly reduce KOA inflammatory pain,delay the progression of KOA subchondral bone disease but may accelerate cartilage degeneration,and it can be used together with Bushen Yiqi recipe rather than using MCC950 or Bushen alone,which had a greater effect on KOA inflammatory pain and structural progression.4.Through cell experiments,it was found the supernatant of Mac cells activated by LPS can stimulate the inflammatory response of FLS,and the medicated serum of Bushen Yiqi Recipe and MCC950 can reduce the inflammatory response by regulating the expression of NLRP3 inflammasome in Mac and FLS.Moreover,the efficiency of MCC950 in inhibiting the NLRP3 inflammasome is significantly higher,the interactions among the groups are complex,and further experiments are needed to optimize and verify that.In conclusion,in vivo and in vitro experiments indicated that Bushen Yiqi recipe could reduce synovial inflammation in KOA guinea pigs with kidney deficiency by regulating the NLRP3 inflammasome. |
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