Analysis Of Syndromes In The Stage Of Bone Loss In Postmenopausal Women And Study On The Mechanism Of CLCF1,the Associated Gene Of Kidney-yin Deficiency Syndrome In Postmenopausal Osteoporosis

Objectives1.To analyze TCM syndromes of postmenopausal women at the stage of bone loss and at all ages.2.To further verify the correlation between CLCF1 and PMOP kidney-yin deficiency syndrome by comparing the expression differences of CLCF1 in postmenopausal women grouped according to TCM syndrome differentiation,bone loss stage,and fracture history.3.To explore the influence of CLCF1 on the OPG/RANKL/RANK system.Methods1.Through a retrospective study of 2823 cases of bone loss stage data collected from2007 to 2020,the symptoms,syndromes,and accompanied symptoms of postmenopausal women in Fujian Province at the stage of bone loss were analyzed.2.By combining the diagnosis of osteoporosis with TCM syndrome differentiation,subjects were divided into groups.The m RNA and protein levels of CLCF1 in peripheral blood mononuclear cells were tested to finally compare the differences in the expression of CLCF1 in each group.3.First,the overexpression technology and CRISPR/Cas9 technology were taken to regulate the expression of CLCF1 gene in THP-1 cells,then the expression changes of OPG,RANKL,IFN?,IFN?,and the change of biological behaviors of THP-1 cells were detected.Results1.In all stages,the highest percentage of symptoms was forgetfulness.As the degree of bone loss increased,the proportion of tooth loss and shaking,lower limb cramps,dry mouth and throat,insomnia,fatigue,fatigue,dizziness,tinnitus,dreaminess,cold limbs,chest tightness,and dizziness showed a linearly increasing trend(P<0.05)whereas proportion of limb numbness showed a linearly decreasing trend(P<0.05).At each stage,the highest percentage of the deficient syndromes was kidney deficiency.As the degree of bone loss increased,the proportions of kidney deficiency,heart deficiency,liver deficiency,spleen deficiency,and qi deficiency showed a linearly increasing trend(P<0.05).At each stage,the highest percentage of kidney deficiency combined with other syndromes were heart deficiency and spleen deficiency,which increased with the increment of bone loss.The proportion of kidney deficiency combined with heart deficiency and kidney deficiency combined with spleen deficiency showed a linearly increasing trend(P<0.05).In each age group at all stages,the main symptoms,the deficient syndromes,the syndromes combined with kidney deficiency,and the common syndromes were basically consistent with the overall characteristics of each stage.2.In the groups of TCM syndrome types,the CLCF1 protein level of PMOP kidney-yin deficiency group was significantly lower than none PMOP kidney yin deficiency group and PMOP without kidney yin deficiency group(P<0.05).The CLCF1 protein level of the PMOP kidney-yin deficiency group had a tendency to be lower than that of the control group without PMOP(P>0.05);In the groups of bone loss,the CLCF1 protein level of the osteoporosis group was lower than the normal bone mass group(P<0.05);In the groups of fracture history,the CLCF1 protein level of the fracture group had a tendency to be lower than the non-fracture group(P>0.05).In addition,the CLCF1 protein level was significantly positively correlated with lumbar bone mineral density(r>0,P<0.05),significantly negatively correlated with the occurrence of osteoporosis(r<0,P<0.05),however not significantly negatively correlated with the occurrence of fractures(r<0,P>0.05).3.When CLCF1 was over expressed,the protein expression of OPG increased(P<0.05);the expression of RANKL did not change significantly(P>0.05);the ratio of OPG/RANKL increased significantly(P<0.01);the expression of IFN? and IFN? decreased(P<0.05);the proliferation,migration and invasion ability of THP-1 cells increased(P<0.05).When CLCF1 was knocked out,the expression of OPG decreased(P<0.05);the expression of RANKL did not change significantly(P>0.05),the ratio of OPG/RANKL had a downward trend(P>0.05);and the expression of IFN? and IFN? increased(P<0.05);the proliferation,migration and invasion ability of THP-1 cells decreased(P<0.05),and the early apoptosis rate increased(P<0.05).Conclusions1.Most postmenopausal women had symptoms such as forgetfulness,low back pain,tooth loss,waist and knee weakness,lower limb cramps,etc.in the low bone mass stage,which were related to the syndromes of kidney yin deficiency.Moreover,these symptoms were identical to the main symptoms of the osteoporosis stage and the severe osteoporosis stage.The proportion of the symptoms rose with the increment of bone loss,suggesting that clinical intervention should be made as soon as possible to relieve the symptoms.2.In the stage of bone loss,the deficiency syndromes mainly exhibited as kidney deficiency and liver deficiency,and common clinical syndromes mainly exhibited as kidney yin deficiency,kidney deficiency and blood stasis.Accompanied syndromes of kidney deficiency mainly included liver deficiency,heart deficiency,and spleen deficiency.It is suggested that the syndromes of OP are complicated;the clinical prevention and treatment should focus on liver and kidney;and the treatment of accompanied syndromes such as heart deficiency should be emphasized.3.CLCF1 was the associated gene of PMOP kidney-yin deficiency syndrome.Its protein expression was significantly positively correlated with lumbar bone mineral density,and significantly negatively correlated with the occurrence of osteoporosis.It is expected to be a molecular marker and therapeutic target for PMOP kidney-yin deficiency syndrome.4.The change of CLCF1 expression could affect the OPG/RANKL/RANK system,thereby participating in bone metabolism.It is expected to become a therapeutic target for bone metabolic diseases.