Duhuo Jisheng Decoction Regulates Intracellular Zinc Homeostasis by Enhancing Autophagy via PTEN/Akt/mTOR to Improve Knee Cartilage Degeneration

Background Articular cartilage degeneration as well as cartilage matrix degradation is one of the key pathological changes in the early stage of knee osteoarthritis (KOA). However, currently, there are limited early prevention and treatment options available. Duhuo Jisheng Decoction (DHJSD) is a formula from Bei Ji Qian jin Yao Fang compiled by Sun Simiao in the Tang Dynasty of China. As a complementary therapy, it is widely used to treat early-stage KOA in China, but its mechanism has not been fully elucidated. Objective This study is aiming at investigating the potential role and mechanism of DHJSD in protecting cartilage from degradation. Methods The mechanism of DHJSD in alleviating OA was explored by gene silencing technology combined with a series of functional experiments in primary rat chondrocytes. Next, 25 wistar rats were used to validate the results obtained in vitro. The PTEN, Akt, mTOR, MMP13, Zn, collagen II, autophagy and apoptosis were determined. Results DHJSD reduced the phosphorylation of Akt and mTOR and the expression of zinc, MMP13, Bax and Bcl2. DHJSD increased the level of autophagy and the expression of autophagy proteins LC3 and Beclin1. After silencing PTEN gene, the phosphorylation levels of Akt and mTOR and the effects of Bax, Bcl2, LC3 and Beclin1 were weakened by DHJSD. DHJSD increased the formation of autophagosomes in chondrocytes. Histopathological staining revealed that DHJSD had a protective effect on cartilage. Conclusion DHJSD inhibits Akt/mTOR signaling pathway by targeting PTEN to promote autophagy in chondrocytes, which may be closely to repress the formation of MMP-13 by regulating the level of zinc in chondrocytes.

autophagosome membrane to participate in the formation of autophagosome [13] . Inhibition of this pathway can increase the level of autophagy [14] . Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a typical tumor suppressor gene that inhibits the PI3K/Akt/mTOR growth signaling cascade [15] . This pathway can be regulated through multiple sites [16] , and its dysfunction will lead to the dysregulation of this pathway and other pathways, leading to overgrowth [17] . At present, PTEN protein has been found to have four heterogeneous structures, PTEN-L, PTEN-M, PTEN-N and PTEN-O, among which PTEN-L plays an important role in the regulation of PI3K/Akt/mTOR signaling pathway and autophagy [18,19] . PTEN can directly inhibit the PI3K/Akt/mTOR pathway, thereby increasing the level of autophagy and reducing apoptosis [20] . PTEN also plays an important role in the regulation of PI3K/Akt/mTOR in the metabolism of chondrocytes. Zhang et al. [21] found that miRNA-132 could up-regulate PTEN and inhibit PI3K/Akt signaling pathway to enhance the level of autophagy in chondrocytes. An Gui-feng et al. [22] studied human chondrocytes and found that miR-365-3p negatively regulated PTEN expression to inhibit the proliferation, migration and invasion of articular chondrocytes and promote their apoptosis. Therefore, the effect and mechanism of DHJSD on cartilage in vitro and in vivo were investigated based on PTEN/Akt/mTOR, autophagy and the zinc homeostasis in chondrocytes in this study.

Preparation of DHJSD-serum
This study was approved by the Experimental Animal Ethics Committee of Hospital of Chengdu University of Traditional Chinese Medicine (No.2023DL-005).Twenty adult male wistar rats (198g-220 g) were obtained from Chengdu Dossy Experimental Animals Co. LTD (Sichuan, China). Rats were bred for 1 week in an environment of 24±2°C, with free access to food and water, and a 12-hour cycle of light/darkness was maintained. Rats were randomly divided into two groups: group A and group B, with 10 rats in each group.
The group A received high dose of DHJSD 2 times daily (1.5mL/100g•d) for 1 week. The same protocol was used with the group B, but the DHJSD was replaced with normal saline. One hour after the last administration, blood was collected from the abdominal aorta, and serum was separated by centrifugation, inactivated in a water bath at 56° C for 30 minutes, packaged, and frozen at -80 ° C until use. Then, all rats were euthanized in accordance with the ethical guidelines for animal welfare. See Fig. 1.

PTEN siRNA
Chondrocytes were transfected with a specific PTEN siRNA synthesized by RiboBio(Guangzhou, China). A 1.5 mL centrifuge tube was prepared, and 15 μL siRNA was diluted with 360μL 1X riboFECT TM CP Buffer and gently mixed. Then 36μL riboFECT TM CP Reagent was added, gently blown and mixed, and incubated at room temperature for 15 min to prepare riboFECT TM CP transfection complex with a final siRNA concentration of 50 nmol for later use. The chondrocytes were seeded in a six-well plate. After the cells adhered to the wall, the supernatant in the wells was removed by suction, and the riboFECT TM CP transfection complex was added to an appropriate amount of complete medium without double antibody, mixed, and added to the well plate, and then incubated at 37℃ and 5%CO 2 for further use.

Cell treatment and CCK-8 assay
Cell grouping and treatments were shown in Fig. 2. CCK-8 assay was used to determine the optimal concentration in four different dosages of Ds (0, 5%, 10%, 15%, 20%). CCK-8 kit was used to detect the cell proliferation rate, and the operation was strictly in accordance with the instructions. Rat primary chondrocytes(Rat-icell-s003, icell) (5×10 4 /mL, 100μL/ well) in each group (4 wells in each group) were seeded in 96-well plates (edge wells were filled with sterile PBS) and cultured for 48 hours at 37℃ with 5% CO 2 . After 48h of treatment, the supernatant was aspirated and discarded. CCK-8 reagent was diluted 1:10 in serum-free medium, and 110μL diluted CCK-8 solution/well was added. The culture plate was gently shaken several times, and the culture was continued for 2 hours at 37℃ and 5% CO 2 . The absorbance values of each well were measured at a wavelength of 450nm using a microplate reader.

Measurement of cell apoptosis
The cells of each group were collected. After the cells were resuspended in 500μL Binding Buffer, 5μL Annexin V-APC/PI was added to blow gently, and then 5 μl PI was added to mix. The reaction was carried out at room temperature in the dark for 15min, and then detected and analyzed.

Determination of intracellular zinc levels
Sample was placed in the Teflon vessel according to the grouping. Concentrated nitric acid (5 mL) was added.
Perform digestion according to the standard operation steps of microwave digestion machine (Shanghai, China). The ICP-MS/MS (iCAP TQ, ThermoFisher, USA) was applied to determine the level of zinc in chondrocytes. The tuning fluid was used to adjust and optimize the instrument parameters before each experiment to make the sensitivity, resolution and stability meet the requirements, and CCT(He/O 2 mode) was used for analysis. The main working parameters and acquisition conditions of the instrument are as follows [23,24]  The rat OA model was established by the Hulth method under anesthesia of pentobarbital sodium. As showed in Fig. 3, the incision was made from the medial side of right posterior knee joint of the rats after anesthetized intraperitoneally with pentobarbital. The muscles as well as ligaments were separated, and the joint cavity was exposed. The medial collateral ligament, anterior and posterior cruciate ligament, as well as the medial meniscus was were cut off. Then, the incision was sutured. After operation, each rat was intramuscular injection with penicillin (400 000 U/d) for 3 consecutive days, and then kept in the cage.

Animal treatments
The model HD group, model MD group and model LD group were given with the same volume of DHJSD at high, medium and low dose by gavage 2 times daily (1.5mL/100g•d) for 4 weeks from the first day after operation, respectively (Fig 4). The same protocol was used with the sham group and the model control group while the DHJSD was replaced with normal saline. After treatment, all rats were euthanized in accordance with the ethical guidelines for animal welfare. The specimens of blood and knee joints were obtained for further tested.

Histological analysis and macroscopic observation
Hematoxylin-eosin staining, masson staining, immunohistochemical staining and trap staining were used to observe the cartilage morphology under light microscope. The autophagosomes of chondrocytes were observed by transmission electronic microscopy.

Elisa
Quantification of the levels of MMP-13 and IL-1β in each group of serum were performed using the corresponding enzyme-linked immunosorbent assay (ELISA) kit. The absorbance at 450 nm was measured by a microplate reader (SpectraMAX Plus384, Molecular Devices, USA).

Real-time PCR
Total cellular RNA was isolated using animal total RNA isolation kit (RE-03014, Foregene, China) according to the manufacture's instruction. Complementary DNA (cDNA) was reverse-transcribed with PrimeScript-RT reagent kit (RR047A, Takara, Japan).The mRNA levels of Beclin-1, Bax, Bcl-2 and PTEN were detected by RT-PCR with TB Green TM Premix Ex TaqTM Ⅱ(Tli RNaseH Plus) (RR820A, Takara, Japan). The complete gene sequences showed in Table 2 were searched from the National Center for Biotechnology Information (NCBI) database, and the specific primers were designed and screened by Primer Premier software. All primers were designed and synthesized by Sangon Bioengineering Technology(Shanghai, China), and purified by ULTRAPAGE. Data were analyzed by 2 −ΔΔCT method.

Western Blot
The protein levels of PTEN, Akt, p-Akt, mTOR, p-mTOR, Bax, Bcl-2, Beclin-1 and LC3-II/I in chondrocytes were detected by Western blot. Total cellular proteins were extracted using RIPA lysates. The proteins were transferred to PVDF membrane by wet rotation method and blocked at room temperature for 1 h. The primary antibody was incubated overnight and the membrane was washed 3 times with TBST for 5 min each time.
The secondary antibody was incubated at room temperature for 2 h, and the membrane was washed 3 times with TBST for 10 min each time. Then the ECL developer solution was uniformly added to the membrane for exposure development. The bands were scanned by exposure using Tianneng GIS chassis control software V2.0, and the results were expressed as the relative expression of the target protein.

Statistical analyses
SPSS 26 (IBM® SPSS® Statistics) was adopted to analyze the data generated in the charts in this experiment, and figures were made by using GraphPad Prism v9.5.1 (GraphPad Software, Inc.). All data are presented as the mean ± SEM, and all experiments were performed three times. Differences among seven groups were analyzed by one-way analysis of variance followed by Fisher's post hoc test. Differences between two groups were statistically analyzed using an unpaired, two-tailed Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Qualitative analysis of compounds contained in DHJSD-serum
A total of 173 compounds were detected. Substances with a total score of 100, including formula, ontology, reference and RT, were listed as in Table 3.

Effect of DHJSD on the viability of chondrocytes incubated with or without IL-1β
The cytotoxicity of Ds (0, 5%, 10%, 15% and 20%) on rat chondrocytes was determined for 48 hours. The results showed that the cell viability of Ds (10 %) treatment for 48 hours was stronger than that of the other four groups. Chondrocytes viability rate was inhibited by IL-1β (10ng/ml), and this effect was reversed when pretreatment with Ds (10%). However, this effect was significantly attenuated after PTEN gene silencing (Fig.   5). Therefore, Ds (10%) was used for subsequent experimental studies.

Effect of DHJSD on apoptosis of chondrocytes in vivo and vitro
In our study, the effect of IL-1β treatment on chondrocytes apoptosis was further confirmed. Ds significantly reduced the level of apoptosis, while this effect was significantly depressed after PTEN gene silencing (Fig.   6). In addition, Bax and Bcl-2 protein expressions were significantly up-regulated after IL-1β treatment, but the effect was reversed when pretreatment with Ds. Synthesis of specific siRNA PTEN can silence PTEN expression and inhibit the anti-apoptotic effect of Ds (Fig. 7A, 7C, 7D).To further verify this phenomenon, experiments in vivo were performed. The results showed that DHJSD can inhibit the expression of Bax and Bcl-2 proteins in chondrocytes of rats with KOA induced by Hulth method, and there was a certain dose dependence (Fig. 7B, 7E, 7F, 7G, 7H). These results indicated that DHJSD produced a significant antiapoptotic effect, which may be achieved by targeting PTEN.

Effect of DHJSD on the level of autophagy in chondrocytes
As shown in Fig. 8A What's more, transmission electron microscopy showed that after treatment with DHJSD, there was a significant increase in autophagic vesicles in the cytoplasm, which was positively correlated with the dose of DHJSD. These results suggested that DHJSD induced autophagy of chondrocytes, which may be mediated by the targeted regulation of PTEN. Collectedly, it was reasonable to speculate that DHJSD promoted autophagy by targeting PTEN according to the results. . After 4 weeks of pretreatment with different dosages of DHJSD in rats with KOA induced by Hulth method, the morphological and structural changes of cartilage organelle were observed by transmission electron microscope. Sham group: cell morphology, mitochondria morphology, rough endoplasmic reticulum morphology and structure were normal. In the model group, chondrocytes were necrotic, the cell body shrank and became smaller, the cell membrane was discontinuous, the cytoplasm was concentrated, the cytoplasmic content was lost in some areas, the nucleus was lysed, and the organelles were dissolved and blurred. In the Model HD group, the morphological structure of cells, the morphological structure of mitochondria were normal, the rough endoplasmic reticulum of some cells was dilated, and there were more autophagosomes in the cytoplasm.

Effect of DHJSD on zinc homeostasis in chondrocytes
A study showed [4] that DHJSD can down-regulate the level of zinc in chondrocytes and inhibit the expression of MMP-13 and other proteins. To verify the effect of DHJSD on intracellular zinc level, ICP-MS/MS was used to measure the level of zinc in chondrocytes in this study. The results showed that the level of zinc in chondrocytes increased after IL-1β treatment. However, Ds reduced intracellular zinc level significantly, and this effect was significantly receded after PTEN gene silencing (Fig. 8K).

Effect of DHJSD on PTEN/Akt/mTOR signaling pathway.
It is well known that the Akt/mTOR signaling axis is closely related to autophagy, and PTEN has a direct regulatory effect on this signaling pathway. In the present study, we found that the pretreatment of Ds promoted PTEN expression and reduced Akt and mTOR phosphorylation in IL-1β-stimulated chondrocytes.
However, when PTEN gene was silenced, the inhibitory effect of Ds on Akt and mTOR phosphorylation was weakened. It is worth mentioning that Ds has no significant effect on PTEN gene, Akt and mTOR phosphorylation levels in normal chondrocytes. Also, the results were confirmed in vivo. See Fig. 9.

DHJSD alleviated cartilage degeneration in OA rat
To observe the chondroprotective effects of DHJSD in vivo, a rat OA model was established by Hulth method.
As shown in Fig. 10, after 4 weeks of Hulth surgery, the model group exhibited severe loss of type II collagen, cartilage destruction, osteoclast formation, and collagen fiber formation, and had a higher Mankin score compared to the sham control group. However, these changes were mild after pre-treatment with a high dose of DHJSD. The results indicated that DHJSD had a certain inhibitory effect on cartilage destruction. In the model group, the joint tissue surface exhibited cartilage erosion, with complete loss of the local cartilage layer structure, a high number of bone cell necrosis, reduced type II collagen levels, increased collagen fiber tissue proliferation deposition area, and a higher number of osteoclasts. After pretreatment with a high dose of DHJSD, a mild cartilage erosion was observed on the joint tissue surface (Fig. 9F, **p<0.01 vs Model control), with a small number of cartilage cell necrosis. There was a significant increase in the expression of type II collagen (Fig. 9G, *p<0.05 vs Model control), and a significant reduction in the deposition area of collagen fiber tissue and the number of surrounding osteoclasts compared to the model group (Fig. 9H, *p<0.05, Fig. 9I, ***p<0.001), as demonstrated by the results of this study. (n=3, mean±SD; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns p>0.05).

Effects of DHJSD on MMP13 and IL-1β in rats with KOA
Besides, we investigated the effect of DHJSD on inflammatory factors in rats with KOA induced by Hulth method. As shown in the Fig. 11, the levels of MMP-13 and IL-1β in the serum, as well as the expression area of MMP-13 in the cartilage matrix were increased in four weeks after the Hulth procedure. However, the corresponding indicators showed varying degrees of reduction in rats with KOA pretreated with DHJSD, and the differences were statistically significant. These results further confirm that DHJSD can reduce the expression of MMP-13 in the cartilage matrix and serum of rats with KOA.

Discussion
Inflammatory cytokine cascade activation, chondrocyte apoptosis, imbalance of subchondral bone remodeling, and extracellular matrix degradation are the four major pathological and etiological characteristics of KOA [25] . Subchondral bone remodeling, delaying chondrocyte apoptosis, and inhibiting matrix degradation are the main research directions for studying cartilage degeneration. Currently, non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used and effective drugs in the conventional clinical treatment of KOA [26,27] .
However, the long-term use of these drugs has side effects such as gastrointestinal ulcers and cardiovascular adverse events [28] , which are concerning. Specific treatments for osteoarthritis, such as glucosamine, hyaluronic acid, and platelet-rich plasma injections, are controversial and either have conflicting guidelines or are not recommended owing to lacking in evidence-based medicine for the long-term efficacy [29] . Therefore, in the current situation, the use of natural methods, including traditional Chinese medicine formulas, is one of the important directions for the prevention and treatment of KOA. The DHJSD has a long history of use in treating KOA, but its mechanism of action has not been fully elucidated.
Degenerative factors run through the whole process of OA, and soft tissue mechanical abnormality is one of the initiating factors of OA. Therefore, the classic Hulth method was used to establish the rat KOA model in this study. IL-1β plays a key role in OA pathogenesis, and it is a well-established method for IL-1β to be used to establish an in vitro cellular OA model. In the present study, both the Hulth-established rat KOA model in vivo and IL-1β-induced cell model significantly replicated human OA-like features, and the Hulth-established rat OA-like features were attenuated by DHJSD.
This study aims to investigate the mechanism of DHJSD in the prevention and treatment of KOA in IL-1β- Autophagy is a key important protective mechanism for cells to avoid their own apoptosis [30,31] . Moreover, the PTEN/Akt signaling axis is closely related to autophagy [11,17,32] . After IL-1β treatment, the expression of PTEN, LC3 and Beclin-1 was down-regulated, and the phosphorylation of Akt and mTOR was increased.
Pretreatment of IL-1β-stimulated chondrocytes with Ds increased the expression of PTEN, LC3 and Beclin-1 and decreased the phosphorylation of Akt and mTOR. However, this effect was weakened when PTEN was silenced. Furthermore, results in vivo showed that the expressions of PTEN, LC3 and Beclin-1 in chondrocytes of rats with KOA established by Hulth method treated with high dose DHJSD were significantly higher than those of the model group, while the expressions of p-Akt and p-mTOR were significantly lower than those of the model group. In addition, the formation of autophagosomes in chondrocytes of rats with KOA established by Hulth method treated with high dose DHJSD was significantly higher than that of the model group by transmission electron microscopy. So, it is reasonable to speculate that DHJSD can target PTEN to inhibit Akt/mTOR signaling pathway and promote the level of autophagy and the expression of LC3 and Beclin-1 proteins in chondrocytes.
As is well known, MMP-13 plays an important role in the progression of OA [33][34][35] . Matrix metalloproteinases can be induced in multiple ways, and inflammatory cytokines such as IL-6 can directly induce production of matrix metalloproteinases, which is the pathological basis for the degradation of articular cartilage [36] . This study showed that DHJSD directly inhibited the formation of IL-1β and MMP-13 in vivo, playing a protective role in articular cartilage.
Zinc is closely related to the formation of matrix metalloproteinases [37,38] . In other words, when cells synthesize more matrix metalloproteinases in an inflammatory state, more zinc is needed to enter the cells.
The present study showed that the intracellular zinc level increased after IL-1β treatment of chondrocytes, and the intracellular zinc level decreased after pretreatment with Ds in chondrocytes stimulated with IL-1β.
Therefore, the downregulation of intracellular zinc level by DHJSD may be one of the reasons for inhibiting MMPs formation. So, DHJSD can enhance the level of autophagy in chondrocytes and LC3 expression, meanwhile reduce the level of zinc in chondrocytes. However, this study is not clear about how zinc in cells is excreted outside through autophagy and whether it is related to the cell exocytosis mechanism, which needs further confirmation (Fig. 12). Although this mechanism needs further clarification, it is not difficult to infer that the maintenance of intracellular zinc homeostasis by DHJSD is closely related to its enhancement of cellular autophagy.