| Backgrounds:Secondary hyperparathyroidism(SHPT)is a common long-term complication of Chronic kidney disease(CKD).SHPT can lead to functional injury of multiple organs,among which the most important clinical manifestation is renal osteodystrophy followed by abnormal bone metabolism in patients,which is also called renal bone disease clinically.Patients may present with fractures,bone and muscle pain and ischemic necrosis of bone tissue.In more severe cases,shrinking man syndrome(shortened height)and Sagliker syndrome(lion face,arched leg,clubbing finger)can occur,which seriously affects the quality of life of patients with CKD and is also a key issue that clinicians have paid close attention to in recent years.As the gold standard for the diagnosis of renal osteodystrophy,bone biopsy is difficult to be accepted by the majority of CKD patients in clinical work due to its traumatic nature.Therefore,it is an urgent problem to find a non-invasive,more sensitive marker that can quickly reflect the level of bone metabolism in patients.At the level of molecular biology,exploring the regulatory mechanism of representative biomarkers in abnormal bone metabolism is the direction we need to further study.In view of this problem,based on the analysis of clinical data,we find that Parathyroidectomy(PTX)is often necessary for radical treatment of severe clinical complications of abnormal bone metabolism in patients with SHPT.However,postoperative patients often develop hypocalcemia/bone hunger syndrome to repair this bone metabolic damage.A large number of clinical studies have shown that the level of abnormal bone metabolism before surgery is closely related to the degree of bone hunger after surgery,that is,the more serious the degree of abnormal bone metabolism before surgery,the more obvious bone pain and bone deformity,the more serious the degree of hypocalcemia/bone hunger after surgery.Therefore,we established a Nomogram prediction model through meta-analysis and R language analysis to find clinically relevant laboratory indicators to predict the level of postoperative bone hunger and reveal the degree of abnormal bone metabolism activity in patients with CKD.Through the analysis of clinical data,it is concluded that: Alkaline phosphatase(ALP)and Parathyroid hormone(PTH)levels are laboratory biomarkers in alkaline phosphatase(ALP)and alkaline phosphatase(PTH)levels to evaluate severe bone metabolism in CKD-SHPT patients.In addition,a large number of literature studies have also shown that PTH plays a very important role in the regulation of bone metabolism.A large number of clinical and basic studies have proved its effectiveness in promoting bone formation through the intermittent administration of PTH at small doses.However,the mechanism of abnormal bone metabolism during the continuous administration of PTH at large doses is still unclear.Therefore,by collecting serum samples of PTX patients before and after surgery,we tried to explore protein molecules related to PTH-induced abnormal bone metabolism by proteomics.Finally,we found that Chitinase 3 like protein 1(CHI3L1)was closely related to it.We continue to further verify its biological function through experiments.CHI3L1 is a cartilaginous glycoprotein-39 first discovered in 1993.In recent years,it has been found that CHI3L1 is involved in the occurrence and development of many diseases,including cancer,atherosclerosis,diabetes,asthma and schizophrenia.It plays an important biological role in activating inflammatory response,macrophage differentiation,apoptosis,and tissue damage and repair.However,its expression level,functional characteristics and mechanism in bone metabolism are still unclear.P38/FOXO1 is an important signal in the MAPK family,among which the Forkhead box protein(FOX)family is a very important transcription factor.Its biological characteristics are complex and diverse,and it is involved in a variety of cellular regulatory processes.Including cell metabolism,oxidative stress,DNA repair,cell cycle arrest,apoptosis and autophagy.Previous studies have confirmed that FOX plays an important regulatory role in the development of many diseases(cancer,bone loss,cardiovascular disease,nerve tissue degeneration),but the current experimental research evidence on whether Fox is involved in the regulation mechanism of PTHinduced abnormal bone metabolism is insufficient.This study hopes to clarify the biological role of CHI3L1 in bone metabolism by further studying the biological function and characterization of CHI3L1 in CKD-SHPT and the possible regulatory mechanism through P38/FOXO1 signaling pathway,which may become an important marker of bone metabolic activity.It may also be a new therapeutic target for renal bone disease.Objectives:1.Investigation of biomarkers for chronic kidney disease-related SHPT metabolic bone disorder through clinical data analysis.2.Illustration of bone metabolism relevant proteins by comparing the differences in proteomics and Parallel Reaction Monitoring(PRM)before and after PTX.3.Characterization of pro-apoptotic role of CHI3L1 in the presence of PTH.4.Demonstration of the mechanistic role of CHI3L1 in the P38/FOXO1 signaling pathway in the context of chronic kidney disease-related metabolic bone disorder.Mthods:(1)Clinical analysis of abnormal bone metabolism in CKD-SHPTClinical and biochemical data of patients with CKD-SHPT maintenance hemodialysis combined with PTX in the Second Hospital of Jilin University from January 2018 to August 2021 were retrospectively collected and analyzed.The Receiver operating characteristic curve(ROC)was drawn to analyze and determine the optimal critical value of influencing factors and test the predictive efficiency of the Logistic regression model.A Nomogram prediction model was established and a line map was drawn,and then internally verified by Bootstrap method(repeated sampling of original data).In addition,we searched Cochrane Library,Embase,Pub Med,Web of Science and other electronic databases,and included a total of 13 studies for meta-analysis to explore their risk factors.It was used to predict the incidence of bone hunger after PTX surgery and to evaluate the level of abnormal bone metabolic activity before surgery.(2)Differential protein expression analysis of abnormal bone metabolism in CKD-SHPTSerum samples of patients undergoing PTX operation before and after operation were collected and divided into groups.Serum samples of patients with PTH >4000pg/ml were selected as pre group in preoperative group,and serum samples of patients on the first day after operation were selected as Pos＿d1 group.In order to prevent the interference of bone metabolism results due to the release of a large number of inflammatory cytokines caused by surgical intervention on the first day after surgery,serum samples of patients with PTX on the seventh day after surgery were collected as Pos＿d7 group.A total of 22 serum samples of patients from the three groups were analyzed for differential expression of proteomics,and the results of analysis were verified by reverse PRM.(3)CHI3L1 was highly expressed in SHPT parathyroid tissue and blood samplesThe expression level of CHI3L1 in parathyroid tissues removed after the operation of PTX patients and in blood samples were detected by various methods.(4)CHI3L1 was highly expressed in PTH-induced osteoblast MC3T3-E1Mouse embryonic osteoblast precursor cells(MC3T3-E1)were cultured for osteogenic differentiation induction,and their osteogenic activity was detected by alizarin red staining,alkaline phosphatase staining,q RT-PCR and Western blot.After osteoblasts were stimulated by PTH with different concentrations and different administration times,the effects of PTH on proliferation capacity of osteoblasts were detected by Cell Counting Kit 8(CCK-8),and the effects of PTH on apoptosis of osteoblasts were detected by flow cytometry and Tunel staining.The expression level of CHI3L1 after PTH stimulation was detected by q RT-PCR and Western blot.(5)CHI3L1 promoted PTH-induced apoptosis of MC3T3-E1 osteoblastsOverexpression of MC3T3-E1 on CHI3L1 at the gene level-lentivirus infection and knock-out-lentivirus infection,Stable overexpressed-lentivirus cell line(OECHI3L1),overexpressed negative control-Lentivirus cell line(OE-CHI3L1-NC),knockout-Lentivirus cell line(KD-CHI3L1)and knockout negative control--Lentivirus cell line(KD-CHI3L1-NC)were formed.The lentivirus-infected cell lines were screened for azathioprine resistance,and the stable strains were tested for GFP fluorescence.At the same time,the expression level of CHI3L1 was detected by q RTPCR and Western blot to evaluate the infectivity of lentivirus.q RT-PCR and Western blot tests were performed on bone metabolism-related proteins and apoptosis-related proteins after large doses of PTH-induced stimulation,to verify the biological effects of CHI3L1 on the osteogenic differentiation and apoptosis induction of PTH-induced MC3T3-E1.(6)Through P38/FOXO1 signaling,CHI3L1 induces apoptosis in MC3T3-E1 osteoblasts.Western blot was used to detect the expression levels of p-P38/P38 and FOXO1 signaling pathway proteins in PTH-induced apoptosis of MC3T3-E1 cells,and to evaluate the activation of P38/ FOXO1 signaling pathway in PTH-induced apoptosis of MC3T3-E1 cells.The expression level of P38/ FOXO1 signaling pathway protein in OE-CHI3L1 and KD-CHI3L1 groups after PTH induction was detected by the same method.After P38-MAPK inhibitor was applied to inhibit the downstream signaling pathway of P38/ FOXO1,the expression levels of pathway-related proteins and apoptosis-related proteins were detected by Western blot,and the apoptosis-related proteins were detected by Tunel staining and flow cytometry.To evaluate the effect of P38-MAPK inhibitors on PTH-induced apoptosis and the regulatory mechanism of CHI3L1 in the P38/ FOXO1 signaling pathway.Results(1)Clinical analysis of abnormal bone metabolism in CKD-SHPTThrough R language analysis of single-center data and meta-analysis of 13 multicenter studies,we finally concluded that preoperative ALP and PTH levels are independent risk factors for hypocalcemia/bone hunger after PTX surgery.On the one hand,it can be used as a good predictor;on the other hand,it also reveals the abnormal active level of bone metabolism in patients before surgery,which provides important clues for us to further study the mechanism.(2)Differential protein expression analysis of abnormal bone metabolism in CKD-SHPTThrough R language analysis of single-center data and meta-analysis of 13 multicenter studies,we finally concluded that preoperative ALP and PTH levels are independent risk factors for hypocalcemia/bone hunger after PTX surgery.On the one hand,it can be used as a good predictor;on the other hand,it also reveals the abnormal active level of bone metabolism in patients before surgery,which provides important clues for us to further study the mechanism.(3)CHI3L1 was highly expressed in SHPT parathyroid tissue and blood samplesThrough the detection of blood samples of patients undergoing PTX surgery and hyperplastic parathyroid tissue,it was found that the expression level of CHI3L1 in blood samples and tissue samples of patients with SHPT was positively correlated with the level of PTH in patients.(4)CHI3L1 was highly expressed in PTH-induced osteoblast MC3T3-E1The biological characteristics and CHI3L1 expression of mouse osteoblasts were observed when different concentrations of PTH were continuously stimulated.1.The realationship of cell cell viability: In the early stage of MC3T3-E1 stimulated by different concentrations of PTH,PTH administration group could enhance cell viability compared with control group.After 48 hours of continuous administration,compared with the control group,cell vitality was inhibited to varying degrees in the PTH continuous administration group,and the degree of inhibition was proportional to the PTH concentration.2.The realationship of osteoclast and osteoblast: On the stimulation of osteoblasts by different concentrations of PTH,the expression of Runt-related transcription factor2(Runx-2)was gradually increased at various time points,and was positively correlated with the concentration of PTH.The expression inflection point of Osteoprotegerin(OPG)and Osteocalcin(OC)was observed when the concentration of PTH was500ng/ml.When the concentration of PTH reached 500ng/ml,the expression level of PTH changed from increasing gradually to decreasing gradually.Correspondingly,the expression of Receptor activator of nuclear factor-κB ligand(Rankl)changed from a gradual decrease to a gradual increase.It was suggested that osteoclast activity began to become active when PTH 500ng/ml was treated for 48 h.3.The realationship of apoptosis: The expression of B-cell lymphocytoma-2(Bcl-2)was observed at 5000 ng/ml after 48 h of continuous PTH administration.For PTH >5000ng/ml,the expression level of Bcl-2 changed from increasing to gradually decreasing after 48 h of continuous administration.Correspondingly,the expression level of the proapoptotic factor Bcl-2 associated X(Bax)changed from a gradual decrease to a gradual increase.It was suggested that the apoptotic activity of osteoblasts was gradually active when PTH was treated for 48 h at 5000ng/ml.Tunel staining and flow cytometry also confirmed this view.4.The expression of CHI3L1: The expression of CHI3L1 reached the optimal value in 5000ng/ml osteoblasts treated with PTH for 48 h.(5)CHI3L1 promoted PTH-induced apoptosis of MC3T3-E1 osteoblasts1.The expression level of CHI3L1 was detected by q RT-PCR and Western blot in the constructed stable strain of overexpressing CHI3L1(OE-CHI3L1)and silenced CHI3L1(KD-CHI3L1),suggesting that CHI3L1 lentivirus successfully infected mouse osteoblasts.2.The cell cycle was detected by flow cytometry and KD-CHI3L1 was blocked in S phase.3.When PTH was treated at 5000ng/ml for 48 h,OE-CHI3L1/KD-CHI3L1 showed a trend of promoting the expression of osteogenic factors Runx-2 and OC compared with the control group.Compared with the control group,OE-CHI3L1 osteoprotectin(OPG)expression was significantly reduced,Rankl expression was gradually increased.Apoptosis-related proteins Bax and Caspase3 were significantly increased,and antiapoptosis-related protein Bcl-2 was significantly decreased.The OE-CHI3L1 group had significantly increased apoptosis compared to the control group based on Tunel staining and flow cytometry.In comparison with the control group,KD-CHI3L1 showed significantly reduced apoptosis.(6)CHI3L1 promoted PTH-induced apoptosis of MC3T3-E1 cells through the P38/FOXO1 signaling pathway.1.P38/FOXO1 pathway signals were continuously administered with large doses of PTH to induce apoptosis for 48 h,and the results of Western blot showed that the relative expression levels of p-P38/P38 and FOXO1 were gradually increased in a dosedependent manner compared with the control group.2.Compared with Ctrl+PTH group,the relative expression levels of p-P38/P38 and FOXO1 in OE-CHI3L1+PTH group were increased,while p-P38/P38 and FOXO1 were significantly decreased in KD-CHI3L1+PTH group.After adding the P38-MAPK inhibitor(SB-203580),The relative expression of FOXO1 in Ctrl+PTH+SB,OECHI3L1+PTH+SB and KD-CHI3L1+PTH+SB groups decreased compared with Ctrl+PTH,OE-CHI3L1+PTH and KD-CHI3L1+PTH groups,respectively.It indicated that P38/FOXO1 downstream signaling pathway was inhibited.After adding P38-MAPK inhibitor(SB-203580),Ctrl+PTH+SB,OE-CHI3L1+PTH+SB and KDCHI3L1+PTH+SB groups were compared with Ctrl+PTH,OE-CHI3L1+PTH and KDCHI3L1+PTH groups.Flow cytometry and Tunel staining showed that the proportion of apoptotic cells decreased significantly.Western blot results showed that the relative expression levels of Bax and Caspase-3 in the inhibitor group were significantly decreased compared with the corresponding control group,while the relative expression levels of Bcl-2 were increased.These results suggest that P38-MAPK inhibitors can inhibit the activation of P38/FOXO1 signaling pathway,thereby reducing PTH-induced apoptosis of MC3T3-E1 cells infected with CHI3L1 lentivirus.Conclusion:1.Preoperative PTH and ALP levels in CKD-SHPT patients are independent risk factors for hypocalcemia after PTX surgery,and can be used as an important indicator to evaluate the degree of abnormal bone metabolism.2.When large dose of PTH continuously stimulated osteoblasts,osteoclast behavior became more active and apoptosis occurred.3.As a differentially expressed protein before and after PTX,CHI3L1 promoted PTH-induced osteoblast apoptosis;4.CHI3L1 promotes PTH-induced osteoblast apoptosis by activating the P38/FOXO1 signaling pathway. |
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