Impacts of cryptotanshinone on autophagy and apoptosis of chondrocytes in rabbit model of knee osteoarthritis by regulating HIF-1α/BNIP3 signaling pathway

doi:10.11958/20230861

Abstract

Abstract:

Objective To investigate the impact of cryptotanshinone on autophagy and apoptosis of chondrocytes in a rabbit knee osteoarthritis (KOA) model by regulating the hypoxia inducible factor-1α (HIF-1α)/BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3) signaling pathway. Methods New Zealand rabbits were selected to construct the rabbit KOA model using the improved Videman method. Rabbits were randomly divided into the model group, the empty group, the cryptotanshinone group, the HIF-1α knockdown group and the cryptotanshinone+HIF-1α knockdown group, with 9 rabbits in each group. Another 9 New Zealand rabbits were taken as the control group. After grouping and intervention, Lequesne MG knee joint level assessment method was used to score clinical symptoms of knee joints (local pain, gait, joint activity and joint swelling) of rabbits. HE staining was used to detect the cartilage degeneration of rabbit knee joint cartilage tissue, and modified Mankin's score was performed. TUNEL staining was used to detect the apoptosis of articular cartilage histiocytes of rabbits. Enzyme-linked immunosorbent assay (ELISA) method was used to detect serum levels of inflammatory cytokines interleukin-6 (IL-6), IL-18 and IL-10 in rabbits. Western blot experiment was used to detect the expression of autophagy (LC3, Beclin-1), apoptosis (Bax, Cleaved Caspase-3) and HIF-1α/BNIP3 signaling pathway related proteins in knee cartilage tissue of rabbits. Results Compared with the control group, the knee joint cartilage tissue of rabbits showed obvious degenerative symptoms, the scores of local pain, gait, joint activity and joint swelling, the modified Mankin's score, apoptosis rate, levels of serum IL-18 and IL-6, and the expression of LC3Ⅱ/LC3Ⅰ and Beclin-1, Bax, Cleaved Caspase-3, BNIP3 proteins in cartilage tissue were increased in the model group, and the serum level of IL-10 and the expression of HIF-1α protein in cartilage tissue were decreased (P<0.05). Compared with the model group, the symptoms of cartilage tissue degeneration in knee joint of rabbits were alleviated, scores of local pain, gait, joint activity and joint swelling, the modified Mankin's score, apoptosis rate, serum levels of IL-18 and IL-6, and the expression of LC3Ⅱ/LC3Ⅰand Beclin-1, Bax, Cleaved Caspase-3, BNIP3 proteins in cartilage tissue were decreased in the cryptotanshinone group, and the serum level of IL-10 and the expression of HIF-1α protein in cartilage tissue were obviously increased (P<0.05). There was no obvious changes in all indicators of rabbits in the empty group (P>0.05). Compared with the cryptotanshinone group, the symptoms of cartilage tissue degeneration in knee joint of rabbits worsened in the cryptotanshinone+HIF-1α knockdown group, and scores of local pain, gait, joint activity and joint swelling, the modified Mankin's score, apoptosis rate, serum levels of IL-18 and IL-6 and the expression of LC3Ⅱ/LC3Ⅰand Beclin-1, Bax, Cleaved Caspase-3, BNIP3 proteins in cartilage tissue were increased, the level of serum IL-10 and the expression of HIF-1α protein in cartilage tissue were decreased (P<0.05). Changes were opposite to those of the HIF-1α knock-down group. Conclusion Cryptotanshinone can up-regulate HIF-1α to down-regulate BNIP3 expression, inhibit inflammation and autophagy, reduce the degeneration of knee joint cartilage tissue in KOA rabbits, and improve its clinical symptoms.

Key words: cryptotanshinone, osteoarthritis, knee, chondrocytes, autophagy, apoptosis, HIF-1α/BNIP3

CLC Number:

R285.5

Figures/Tables 9

References 21

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组别	局部疼痛	步态	关节活动	关节肿胀
对照组	0（0，0）	0（0，0）	0（0，0）	0（0，0）
模型组	2（2，2）^a	2（2，2）^a	2（2，2）^a	1（1，1）^a
空载组	2（2，2）	2（2，2）	2（2，2）	1（1，1）
隐丹参酮组	0（0，1）^b	0（0，1）^b	0（0，1）^b	0（0，0）^b
HIF-1α敲低组	3（2，3）^b	3（3，3）^b	3（2，3）^b	2（2，2）^b
隐丹参酮+ HIF-1α敲低组	2（1，2）^cd	2（1，2）^cd	1（1，2）^cd	1（1，1）^cd
H	125.441^＊	135.052^＊	172.203^＊	109.246^＊

组别	局部疼痛	步态	关节活动	关节肿胀
对照组	0（0，0）	0（0，0）	0（0，0）	0（0，0）
模型组	2（2，2）^a	2（2，2）^a	2（2，2）^a	1（1，1）^a
空载组	2（2，2）	2（2，2）	2（2，2）	1（1，1）
隐丹参酮组	0（0，1）^b	0（0，1）^b	0（0，1）^b	0（0，0）^b
HIF-1α敲低组	3（2，3）^b	3（3，3）^b	3（2，3）^b	2（2，2）^b
隐丹参酮+ HIF-1α敲低组	2（1，2）^cd	2（1，2）^cd	1（1，2）^cd	1（1，1）^cd
H	125.441^＊	135.052^＊	172.203^＊	109.246^＊

组别	改良Mankin's评分/分	凋亡率/%
对照组	0（0，0）	1.14±0.33
模型组	3（3，3）^a	38.26±5.31^a
空载组	3（3，3）	39.13±5.46
隐丹参酮组	1（0，1）^b	3.45±0.62^b
HIF-1α敲低组	4（4，4）^b	56.74±8.10^b
隐丹参酮+HIF-1α敲低组	2（2，3）^cd	34.83±6.05^cd
H或F	145.365^＊	162.700^＊

组别	改良Mankin's评分/分	凋亡率/%
对照组	0（0，0）	1.14±0.33
模型组	3（3，3）^a	38.26±5.31^a
空载组	3（3，3）	39.13±5.46
隐丹参酮组	1（0，1）^b	3.45±0.62^b
HIF-1α敲低组	4（4，4）^b	56.74±8.10^b
隐丹参酮+HIF-1α敲低组	2（2，3）^cd	34.83±6.05^cd
H或F	145.365^＊	162.700^＊

组别	IL-18	IL-6	IL-10
对照组	310.25±34.32	126.18±21.32	52.33±9.21
模型组	984.63±89.57^a	453.84±57.19^a	23.86±3.57^a
空载组	958.56±92.43	464.53±62.45	21.02±4.13
隐丹参酮组	332.42±38.21^b	141.25±25.21^b	45.11±9.42^b
HIF-1α敲低组	1 564.82±162.55^b	821.39±86.53^b	3.45±0.64^b
隐丹参酮+ HIF-1α敲低组	856.71±91.39^cd	430.72±60.47^cd	25.24±3.96^cd
F	219.800^＊	182.100^＊	76.740^＊