Triptolide Inhibits the Activation of NLRP3 Inammasome by Inhibiting NF-κB Pathway and Improves Myocardial Fibrosis

Objective Triptolide (TPL) is identied to be involved in the treatment for myocardial brosis (MF). This study investigated the mechanism of TPL in MF in rats and observed its effect on NLRP3 inammasome signaling pathway. The MF rat model was established by subcutaneous injection of isoproterenol (ISO), and treated by subcutaneous injection of TPL. After modeling for 1 week, the cardiac function of rats was evaluated, including LVEF, LVFS, LVES, LVED LVIDs and LVPWs. The HMI and LVMI were measured. The expressions of ANP, BNP, inammatory related factors (IL-1β, IL-18, TNF-α, MCP-1, VCAM-1), and NLRP3 inammasome factors (NLRP3, ASC, caspase-1) in rats were detected. HE staining and Masson staining were used to observe myocardial cell inammation and brosis of rats. LVED, LVES, LVIDs and LVPWs of MF group were signicantly upregulated, LVEF and LVFS were signicantly downregulated, HMI and LVMI were upregulated, while TPL treatment reversed these trends; TPL treatment downregulated the tissue injury and improved the pathological damage of MF rats. TPL treatment downregulated the levels of inammatory factors and brosis factors, and inhibited the activation of NLRP3 inammasome. Activation of NLRP3 inammasome or NF-κB pathway reversed the effect of TPL on MF. The collagen volume fraction of rats MF and TPL group reduced CVF of rats signicantly. The tissue injury in MF rats upregulated signicantly while that in TPL group was decreased effectively, indicating that TPL could pathological damage of MF rats. TPL inhibits collagen gel and suppresses the production of A study has shown can alleviation effect of TPL on the diabetic renal brosis by inhibiting miR-141-3p/PTEN/Akt/mTOR pathway This study explored the role of TPL in alleviating the brosis and the expression of brosis-related factors. The brosis related factors (TGF-β1, COL1 and COL3) expressions in MF and DMSO groups were much higher, while those in TPL group were much lower. Consistently, TPL inhibits smooth muscle cells malignant suggested that TPL could MF and downregulate factors


Introduction
Myocardial brosis (MF) is de ned as various quantitative and qualitative changes of the myocardial interstitial collagen network which occurs in response of cardiac ischemic injuries, systemic diseases, drugs, or any other harmful stimulation affecting the circulatory system or the heart itself [1]. MF is a common phenomenon in the late stage of many heart diseases and is a predictor of sudden cardiac death [2], which may be caused by a series of pathological processes [3]. Myocardial broblasts become pro-in ammatory cells and activating the in ammasome in the early stages of infarct healing [4]. Evidence also supports the effect of in ammation on the initiation and promotion of diverse cardiovascular diseases, including MF [5]. MF changes the myocardium architecture, leads to cardiac dysfunction, arrhythmias and ischemia, and thus in uences the clinical course and prognosis of patients with heart failure [6]. However, there still lacks satisfactory treatment for MF and further work is needed for the effective intervention of MF.

Triptolide (TPL) is a main active component extracted from traditional Chinese herbal medicine
Tripterygium wilfordii, which exhibits multiple pharmacological effects [7], including potent antitumor, immunosuppressive, and anti-in ammatory properties, but the effects are limited in clinic because of the poor solubility, toxicity and bioavailability [8]. The value of TPL in the treatment of various in ammatory disorders and cancers has been emphasized over the past few years [9,10]. Evidence has also shown that the TPL plays a role in attenuating cardiac brosis [11]. In ammatory is one of the best-studied mediators related to cardiac brosis [12]. The NLRP3 in ammasome is noticed as a key to regulate metabolic in ammation [13]. The inhibitor of NLRP3 can alleviate the MF [14]. It is reported that an appropriate dose of TPL can inhibit the activation of NLRP3 in ammasome [15]. It is shown that TPL can downregulate NLRP3 [16]. Collectively, TPL may play an effective role in interrupting the activation of NLRP3 in ammasome to improve MF.
At present, there are few reports on the effect of TPL on treating MF induced by isoproterenol (ISO), and the mechanism of TPL improving the condition of MF needs to be further explored. This study investigated the protective mechanism of TPL on MF rats, and shall provide a new theoretical basis for the improvement of TPL on MF.

Ethics statement
All procedures were authorized by the academic ethics committee of The Central Hospital A liated to Shaoxing University (ethics committee approved number: 2019-003). All procedures were strictly implemented by the guidelines of Animal Care Committee of The Central Hospital A liated to Shaoxing University. All the laboratory procedures were used to reduce the pain of the rats, such as heating pads, disinfection and replenishing uids with saline.

Laboratory animals and treatment
Adult male Sprague-Dawley (SD) rats, 8-10 weeks old, weighing 220-270 g, were purchased from Shanghai Shrek experimental animal Co., Ltd (Shanghai, China). The rats were raised in a clean room in a 12-h light/dark cycle at 20℃ with 50% and 60% humidity. All animals were euthanized by intraperitoneal injection of pentobarbital sodium ≥ 100 mg/kg.
All the 42 SD rats were randomly assigned into 7 equal groups, with 4 groups for studying the effect of TPL on MF: sham group (subcutaneously injected with 5 mg/kg/d saline), myocardial brosis (MF) group (subcutaneously injected with 5 mg/kg/d isoproterenol, ISO), dimethyl sulfoxide (DMSO) group (subcutaneously injected with ISO at 5 mg/kg/d and DMSO at 200 µg/kg/d) and triptolide (TPL) group (subcutaneously injected with ISO at 5 mg/kg/d and intraperitoneal injected with TPL at 200 µg/kg/d, which was dissolved in DMSO) (Sigma, St Louis, MO, USA) for 7 d [17]. The heart function was measured 7 d after injection and following experiments were carried out. The other 3 groups were used for the joint experiment to study the effect of TPL on MF through NLRP3 in ammasome and NF-κB: TPL + PBS group, TPL + Nigericin group and TPL + Jaceosidin group. The rats in TPL + Nigericin and TPL + Jaceosidin groups were intraperitoneally injected with NLRP3 in ammasome activator Nigericin (2.5 mg/kg/d) (Sigma) or NF-κB pathway activator Jaceosidin (2.5 mg/kg/d) (Sigma) for 7 d [18,19], and the injection of PBS was used as the control group.

Measurement of heart mass index (HMI) and left ventricular mass index (LVMI)
All animals were euthanized by an intraperitoneal injection of pentobarbital sodium ≥ 100 mg/kg after cardiac function test [20]. The hearts of rats were collected and the large blood vessels and adipose tissues were removed. The hearts were washed with PBS for 3 times, dried with lter paper, and weighed.
The left ventricle was separated and weighed. HMI was calculated by heart weight/rat body weight, and LVMI was calculated by left ventricular weight/rat body weight.

Sample collection
All animals were euthanized by an intraperitoneal injection of pentobarbital sodium ≥ 100 mg/kg after cardiac function test [20]. The myocardial tissues of some rats were taken out and embedded in para n and each specimen was cut into sections at 4 µm for histological experiments. The myocardial tissues of the rest rats were made into homogenate for subsequent detection.

Hematoxylin and eosin (HE) staining
The rat hearts were soaked in 4% paraformaldehyde for 24 h, and each specimen was cut into sections at 4 µm. HE staining was performed after dewaxing. The sections were stained with hematoxylin for 5 min and stained with eosin for 5 s. Then sections were then dehydrated, cleared, and sealed by neutral resin and nally the pathological damage of rats was observed by microscope.

Masson staining
The prepared myocardial tissue sections were treated with Masson staining solution (Sigma), 1% phosphotungstic acid solution, aniline blue solution, and 1% glacial acetic acid solution successively, and images were acquired and analyzed after dehydration and clearance. The collagen bers were stained blue, the nucleus was stained blue, and the cytoplasm was stained red. Blind method was used to analyze 5 visual elds in each section and the blue area of collagen bers. The collagen volume fraction (CVF) was measured. CVF = the blue area of collagen bers/full eld area × 100%, and the mean value was calculate.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) Total RNA of myocardial tissue homogenate was respectively extracted by RNAiso Plus (TAKARA, Otsu, Shiga, Japan) and Trizol LS Regent (TAKARA). The reliability of extracted RNA was veri ed by formaldehyde denaturation electrophoresis. Subsequently, the reverse transcription experiment was performed with PrimeScript™ RT kit (TAKARA) in strict accordance with the instructions. SYBR Premix Ex Taq (TAKARA) was used to quantify the relative expression of genes by RT-qPCR. GAPDH was used as reference gene. The primers in each group were shown in Table 1.

TPL improved cardiac dysfunction in MF rats
TPL is the main active component of tripterygium wilfordii hook, which has been reported for many times that for the functions of anti-in ammation, anti-tumor and immunosuppression [9]. However, the role of TPL in MF rats remains to be further studied. One week after ISO induction, the cardiac function of rats was measured by small animal ultrasonograph. Compared with sham-operated rats, LVED LVES, LVIDs and LVPWs of rats in MF group and DMSO group were signi cantly upregulated, while LVEF (%) and LVFS (%) were signi cantly decreased, which indicated that the systolic and diastolic functions of rat hearts induced by ISO were signi cantly impaired, and cardiac dysfunction existed, while TPL treatment effectively improved the cardiac dysfunction of MF rats (all p < 0.05, Fig. 1A-F). The rat heart tissues were collected. Compared with sham group, the HMI, and especially the LVMI in MF group and DMSO group were signi cantly increased, while TPL treatment signi cantly improved the increase of HMI and LVMI induced by ISO (Fig. 1G). Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are secreted by ventricular myocytes, and their concentrations are closely related to cardiac function [21,9]. Therefore, the mRNA expressions of ANP and BNP in rat myocardium were measured. The results showed that the levels of ANP and BNP in MF group and DMSO group were higher than those in sham group, while the levels of ANP and BNP in TPL group were signi cantly decreased (all p < 0.01, Fig. 1H). These results suggested that TPL could effectively improve cardiac dysfunction in MF rats.
TPL reduced the degree of brosis in rats MF could cause myocardial tissue disorder and large amount of collagen deposition [22]. Therefore, HE staining and Masson staining were used to observe the pathological morphology and MF of rats in each group. HE staining showed that compared with sham group, the disorder of myocardial tissue structure and injury of MF group and DMSO group were signi cantly aggravated, while the tissue injury degree of TPL group was effectively decreased (p < 0.01), which indicated that TPL treatment could improve degree of the pathological damage of MF rats. Masson staining demonstrated that there were abundant collagen bers in the rats of MF group and DMSO group ( Fig. 2A). CVF quantitative analysis indicated that the collagen volume fraction of rats in MF group and DMSO group was signi cantly increased relative to that of sham group, and TPL treatment signi cantly reduced CVF of rats (p < 0.01, Fig. 2B). RT-qPCR was used to detect the expression of brosis related factors. Expressions of brosis related factors TGF-β1, COL1 and COL3 in MF group and DMSO group were higher than that in sham group, while those in TPL group were lower than that in MF group (all p < 0.01, Fig. 2C). These results suggested that TPL could downregulate the degree of brosis in rats.

TPL downregulated the level of in ammatory factors in MF rats
Cardiac dysfunction was often accompanied by in ammation [23,24]. The mRNA and protein levels of in ammatory factors IL-1β, IL-18 and TNF-α in myocardial tissue of rats were detected in each group. Compared with sham group, the mRNA levels of in ammatory factors IL-1β, IL-18 and TNF-α in MF group and DMSO group were signi cantly upregulated, while TPL treatment effectively decreased the expression of in ammatory factors (all p < 0.01, Fig. 3A). Monocyte chemoattractant protein 1 (MCP-1) and vascular cell adhesion molecule 1 (VCAM-1) are also key in ammatory factors, which could promote the occurrence of in ammatory reaction [25,26]. The mRNA expressions of MCP-1 and VCAM-1 were also detected, and the results were consistent with our predicted trend. TPL treatment could downregulate the expression of MCP-1 and VCAM-1 in MF rats (all p < 0.01, Fig. 3B). These results suggested that TPL could downregulate the in ammatory level of MF rats.

TPL inhibited the activation of NLRP3 in ammasome in MF rats
Subsequently, RT-qPCR and WB were used to respectively detect the main components of NLRP3 in ammasome mRNA and protein levels of NLRP3 and ASC. Compared with sham group, the mRNA and protein levels of NLRP3 and ASC in MF group and DMSO group were signi cantly upregulated, and TPL treatment effectively downregulated their expressions (all p < 0.01, Fig. 4A-D). NLRP3 in ammasome can induce pro-caspase-1 self-cleavage to activated caspase-1 [27]. Therefore, the activation of caspase-1 was detected by WB, and the results also showed that compared with sham group, the expression of caspase-1 in MF group and DMSO group were signi cantly upregulated, while TPL treatment effectively downregulated the expression of caspase-1 (all p < 0.05, Fig. 4E). It was suggested that TPL inhibited the activation of NLRP3 in ammasome in MF rats.

TPL improved brosis degree of MF rats by inhibiting the activation of NLRP3 in ammasome pathway
It was con rmed through previous experiments that TPL could inhibit the in ammasome of NLRP3 and improve the MF. Therefore, we speculated that TPL might improve the MF by regulating the NLRP3 in ammatory pathway. In order to verify the conjecture, a joint experiment was conducted: NLRP3 in ammasome activator Nigericin was added in TPL-treated group, with the addition of PBS as control group. The changes of brosis degree in MF rats were observed. After adding Nigericin, LVED, LVES, LVIDs and LVPWs were signi cantly increased, LVEF and LVFS were signi cantly decreased (Fig. 5A-F), the myocardial tissue structure was disordered, the injury was aggravated, and the volume fraction of collagen was increased ( Fig. 5G-H). Moreover, compared with TPL + PBS group, the mRNA expressions of in ammatory factors IL-1β, IL-18 and TNF-α in TPL + Nigericin group were higher than those in control group (all p < 0.01, Fig. 5I), indicating that NLRP3 pathway was successfully interfered, and the in ammatory level of TPL + Nigericin group was upregulated. Subsequently, the mRNA expressions of brosis related factors (TGF-β1, COL1 and COL3) were detected by RT-qPCR. The mRNA expressions of brosis related factors in TPL + Nigericin group were signi cantly higher than that in TPL + PBS group (all p < 0.01, Fig. 5J). These results suggested that TPL improved the degree of MF in MF rats by inhibiting the activation of NLRP3 in ammasome pathway.
TPL inhibited the activation of NLRP3 in ammasome by inhibiting NF-κB pathway and thus improving the MF in rats NLRP3 pathway is an important downstream pathway of NF-κB pathway, and NF-κB pathway is often reported to be associated with in ammation and MF [28,29]. It was speculated that TPL might inhibit the activation of NLRP3 in ammasome by inhibiting the NF-κB signaling pathway and improve the degree of MF. Therefore, the expression of NF-κB pathway related proteins was detected, and it was found that P65 phosphorylation levels in MF group and DMSO group were higher than that in sham group, while TPL treatment effectively downregulated P65 phosphorylation level (p < 0.01, Fig. 6A). Subsequently, the NF-κB pathway activator Jaceosidin was used to treat rats in the TPL group, and the injection of PBS was used as the control group. It was found that after Jaceosidin treatment, rat cardiac function indexes LVED, LVES, LVIDs and LVPWs were signi cantly increased, LVEF and LVFS were signi cantly decreased (Fig. 6B-G), the improvement of TPL on myocardial tissue structure damage was reversed, myocardial tissue damage was aggravated, and collagen volume fraction was increased (Fig. 6H-I). The changes of in ammatory and brosis related factors in myocardial tissue were detected by RT-qPCR. Levels of in ammatory factors and brosis factors in TPL + Jaceosidin group were higher than those in TPL + PBS group (p < 0.01, Fig. 6J-K). All the above results con rmed that TPL could improve the MF by inhibiting NF-κB pathway and the activation of NLRP3 in ammasome. Discussion MF, mainly caused by the death of ischemic cells in the process of myocardial infarction that leads to a reparative response in which the damaged tissue is replaced with a brotic scar, can lead to impairment of cardiac function and nally causes adverse outcomes of heart failure [30]. However, there is no therapeutic strategy that can ensure its reversal at present [31]. Evidences have shown that TPL has the characteristics of anti-in ammatory and antitumor [9]. This study found that TPL could inhibit the activation of NLRP3 in ammasome and improve MF by inhibiting NF-κB pathway.
This study found that TPL could inhibit the activation of NLRP3 in ammasome and improve MF by inhibiting NF-κB pathway.
TPL has been shown to have potent antitumor activity in various preclinical cancer models [8], such as lung cancer, breast cancer and pancreatic cancer [32][33][34]. However, the effect of TPL on MF was still unclear. MF can contribute to cardiac dysfunction in many pathophysiologic conditions [12]. The levels of LVED, LVES, LVEF and LVFS are main indexes of cardiac function [35]. This study showed that TPL improved cardiac dysfunction in MF rats. LVED, LVES, LVIDs and LVPWs were signi cantly upregulated, while LVEF and LVFS were signi cantly decreased in MF rats, indicating that the systolic and diastolic functions of rat hearts were signi cantly impaired; and the cardiac dysfunction of MF rats was effectively improved by TPL treatment. The concentrations of ANP and BNP are closely associated with cardiac function [21]. The levels of ANP and BNP in MF rats were signi cantly higher, while the levels of ANP and BNP in TPL group were signi cantly decreased. Consistently, it has been revealed that TPL therapy improves cardiac function through upregulation of MAPK signaling transduction [36]. In brief, TPL could effectively improve cardiac dysfunction in MF rats.
MF can cause myocardial tissue disorder and collagen deposition [22]. The collagen bers in the rats of MF and DMSO groups were abundant. The collagen volume fraction of rats in MF and DMSO groups was increased signi cantly, and TPL group reduced CVF of rats signi cantly. The tissue injury in MF rats was upregulated signi cantly while that in TPL group was decreased effectively, indicating that TPL could improve pathological damage of MF rats. TPL inhibits collagen gel and suppresses the production of bronectin [37]. A study has shown can alleviation effect of TPL on the diabetic renal brosis by inhibiting miR-141-3p/PTEN/Akt/mTOR pathway [38]. This study explored the role of TPL in alleviating the brosis and the expression of brosis-related factors. The brosis related factors (TGF-β1, COL1 and COL3) expressions in MF and DMSO groups were much higher, while those in TPL group were much lower. Consistently, TPL inhibits airway smooth muscle cells malignant behaviors induced by TGF-β1 signi cantly [39]. These suggested that TPL could alleviate MF and downregulate the brosis related factors expression in MF rats.
Cardiac dysfunction is often accompanied by in ammation [24]. Our results showed that the levels of in ammatory factors (IL-1β, IL-18 and TNF-α) in MF and DMSO groups were upregulated signi cantly, while TPL treatment decreased the expression of in ammatory factors effectively. It is reported that TPL can downregulate the expressions of IL-1β and TNF-α [40]. TPL attenuates in ammatory responses in endothelial cells [41]. In brief, these results suggested that TPL could downregulate the level of in ammatory in MF rats.NLRP3 in ammasome can nally produce a mature form of highly in ammatory cytokines [42]. The mRNA and protein levels of NLRP3 and ASC were increased signi cantly in MF and DMSO groups, while TPL treatment decreased their levels effectively. It is reported that TPL can inhibit the activation of NLRP3 in ammasome and protect against the in ammatory injury in diabetic kidney disease [15]. We speculate that TPL may improve MF by regulating NLRP3 in ammasome pathway. To test our hypothesis, we added NLRP3 in ammasome activator Nigericin in TPL group. Our results demonstrated that mRNA expressions of in ammatory factors and brosis related factors in TPL + Nigericin group were higher. There is a previous study supporting that TPL protects against pressure overload of hearts by inhibiting the activation of NLRP3 in ammasome [43]. Treatment with TPL exerts the anti-in ammatory effects by downregulating NLRP3 in ammasome in nephritis [44]. In brief, it was suggested that TPL improved MF by inhibiting the activation of NLRP3 in ammasome pathway.
NF-κB pathway is reported to play an essential role in myocardial in ammation as a transcription factor [29]. TPL improves podocyte apoptosis and proteinuria by inhibiting NF-κB/GADD45B pathway [45]. We deduced that TPL might inhibit NLRP3 in ammasome via inhibiting NF-κB pathway. The results showed that P65 phosphorylation levels in MF and DMSO groups were elevated, while TPL treatment downregulated the levels effectively. Next, we treated the TPL group with NF-κB pathway activator Jaceosidin. The levels of in ammatory factor and brosis factors in TPL + Jaceosidin group were signi cantly enhanced. TPL may suppress in ammatory response of endothelial cells by inhibiting activation of NF-κB [41]. There is little research on the effect of TPL on inhibiting MF via NF-κB pathway. Our study con rmed that TPL could improve MF by inhibiting NF-κB pathway and the activation of NLRP3 in ammasome.
In summary, this study supported that TPL could inhibit the activation of NLRP3 in ammasome by inhibiting NF-κB pathway, thus improving MF. Whether there is a deeper regulatory mechanism between TPL and NF-κB pathway in MF remains to be further studied. Our results showed that TPL was only a preventive method and does not necessarily reverse the MF established after ISO injection. Its therapeutic effect needs to be further studied. Studies have shown that TPL can inhibit in ammation [46,41]. There may be some feasibility in the TPL treatment. However, TPL is also the main toxic component, which has serious side effects on many important tissues. The kidney is very vulnerable to the side effects of TPL as an important excretory organ, which greatly limits the clinical application of TPL [47]. Therefore, the research on the toxicity reduction and e ciency enhancement of TPL is of great practical signi cance and needs to be further explored.It is the future research trend to transfer the mechanism of TPL on improving MF to clinical application.

Competing interests
The authors declared that they have no competing interests. and LVED were measured by the small animal ultrasonograph; (E) RT-qPCR was used to detect the ANP and BNP levels in myocardial tissues of rats in each group; N = 6. Three independent repeated tests were performed and the data were expressed as mean ± standard deviation; one-way or two-way ANOVA was used for variance analysis; Tukey's multiple comparisons test was used for post hoc test. Compared with sham group, *p < 0.05, **p < 0.01; compared with MF group, #p < 0.05, ##p < 0.01. (C) RT-qPCR was used to detect the expressions of TGF-β1, COL1 and COL3; (D) The expressions of TGF-β1, COL1 and COL3 were detected by immunohistochemistry; N = 6. Three independent repeated tests were performed and the data were expressed as mean ± standard deviation; one-way or two-way ANOVA was used for variance analysis; Tukey's multiple comparisons test was used for post hoc test. Compared with sham group, *p < 0.05, **p < 0.01; compared with MF group, #p < 0.05, ##p < 0.01.

Figure 3
TPL downregulated the level of in ammatory factors in MF rats. (A-B) RT-qPCR and WB were used to respectively detect the mRNA and protein levels of in ammatory factors IL-1β, IL-18 and TNF-α in the myocardium of rats; (C) RT-qPCR was used to detect the mRNA expression of MCP-1 and VCAM-1 in myocardium of rats in each group; N = 6. Three independent repeated tests were performed and the data were expressed as mean ± standard deviation; two-way ANOVA was used for variance analysis; Tukey's multiple comparisons test was used for post hoc test. Compared with sham group, **p < 0.01; compared with MF group, ##p < 0.01. TPL inhibited the activation of NLRP3 in ammasome in MF rats. (A-D) RT-PCR and WB were used to detect the mRNA and protein levels of NLRP3 and ASC in MF rats; (E) The expressions of NLRP3 and ASC were detected by immunohistochemistry; N = 6. Three independent repeated tests were performed and the data were expressed as mean ± standard deviation; one-way ANOVA was used for variance analysis; Tukey's multiple comparisons test was used for post hoc test. Compared with sham group, **p < 0.01; compared with MF group, ##p < 0.01. TPL improved the degree of MF in MF rats by inhibiting the activation of NLRP3 in ammasome pathway.
(A) RT-qPCR was used to detect the mRNA expressions of in ammatory factors IL-1β, IL-18 and TNF-α in myocardial tissues; (B) RT-qPCR was used to detect the mRNA expressions of TGF-β1, COL1 and COL3; (C) The expressions of TGF-β1, COL1 and COL3 were detected by immunohistochemistry; N = 6. Three independent repeated tests were performed and the data were expressed as mean ± standard deviation; two-way ANOVA was used for variance analysis; Tukey's multiple comparisons test was used for post hoc test. Compared with TPL + PBS group, **p < 0.01.