Summary
Objective: To investigate the therapeutic effects and mechanisms of Tetracycline (Tet) in mouse models of severe acute pancreatitis (SAP) in vivo and in vitro.

Method (1) Extract the pancreas of C57BL/6 mice, isolate primary pancreatic acinar cells (PACs), and randomly divide them into blank control (Ctrl) group, Tet group, injury [palmitoleic acid (POA) or taurocholic acid sodium (TLCS)] group, and Tet treatment (Tet+POA or Tet+TLCS) group. Fluo-4 fluorescent probe was used to detect changes in cytoplasmic calcium concentration; Propidium iodide staining was used to detect the level of cell necrosis; TMRM fluorescent probe is used to detect changes in mitochondrial membrane potential; DCFH-DA fluorescent probe is used to detect levels of reactive oxygen species (ROS). (2) 35 healthy 8-week-old C57BL/6 male mice were randomly divided into Ctrl group, sham operation group, Tet group, SAP model [fatty acid ethyl ester induced acute pancreatitis (FAEE-AP) or TLCS induced acute pancreatitis (TLCS-AP)] group, and Tet treatment (Tet+FAEE-AP or Tet+TLCS-AP) group, with 5 mice in each group. Take pancreatic tissues from each group of models and observe pathological changes through HE staining; Immunohistochemistry was used to observe the positive expression of C/EBP homologous protein (CHOP); Western blot was used to detect the levels of glucose regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, and CHOP proteins. Collect serum and measure serum amylase activity, interleukin-6 (IL-6) levels, and oxidative stress factors such as malondialdehyde (MDA), glutathione peroxidase (GSH Px), superoxide dismutase (SOD), and catalase (CAT) levels. Take pancreatic and lung tissues from mice and detect myeloperoxidase (MPO) activity.

Result (1) In cell experiments, Tet effectively inhibited calcium overload and necrosis induced by POA or TLCS in PACs, alleviated the decrease in mitochondrial membrane potential of PACs, and reduced ROS generation within PACs (P<0.01). (2) In animal experiments, compared with the Ctrl group, the SAP model group showed a significant increase in pancreatic histopathological scores, serum amylase activity, serum IL-6 levels, pancreatic MPO activity, and lung MPO activity (P<0.01); Tet treatment can effectively alleviate the above-mentioned tissue damage and inflammatory response (P<0.01). Compared with the SAP model group, Tet treatment can reduce the expression of endoplasmic reticulum stress-related GRP78/PERK/CHOP signaling pathway (P<0.01), decrease serum peroxide MDA, and increase antioxidant GSH Px, SOD, and CAT (P<0.01).

Conclusion: Tet can inhibit calcium overload, alleviate mitochondrial membrane potential decrease, alleviate endoplasmic reticulum stress response and oxidative stress damage, thereby reducing the necrosis of PACs and exerting a preventive and therapeutic effect on SAP in mice.

Keywords severe acute pancreatitis; Han Fangji Jia Su; Calcium overload; Endoplasmic reticulum stress; oxidative stress

Acute pancreatitis (AP) is a common digestive system disease in clinical practice, caused by multiple pathogenic factors leading to premature activation of pancreatic enzymes and digestion of the pancreas and its surrounding environment [1-3]. In recent years, the incidence rate of AP has continued to rise, and its etiology has regional and ethnic differences [4]. Alcohol abuse, complications of gallstones, and side effects of medication can all cause AP. About 20% to 25% of AP patients progress to severe AP (SAP), with poor prognosis and high mortality rate after onset [5].
During the pathogenesis of acute pancreatitis, the continuously elevated intracellular Ca2+has cytotoxicity, leading to premature activation of digestive enzymes in pancreatic acinar cells (PACs), causing self digestion of pancreatic tissue [6-10]. At the same time, calcium overload stimulates mitochondria to produce excessive reactive oxygen species (ROS), causing cellular dysfunction, mitochondrial damage, endoplasmic reticulum stress (ERS), and cell death [11]. At present, there is still a lack of targeted drugs for the treatment of AP. The use of specific Ca2+channel blockers to alleviate calcium overload in PACs has the potential to develop into a therapeutic strategy for AP.
Tetrandrine (Tet), also known as tetrandrine, is a bisbenzylisoquinoline alkaloid extracted from the plant Tetrandrine in the family Tetrandriaceae. It has multiple pharmacological effects. Tet has shown good activity in anti-inflammatory, anti-tumor, and immune regulation aspects [13]. Meanwhile, the pharmacological action of Tet is manifested as a Ca2+channel antagonist, which can effectively block certain Ca2+channels, such as voltage dependent L-type and T-type Ca2+channels [14]. In recent years, studies have shown that Tet is a specific blocker of two pore channels (TPCs) [15]. In addition, in rat SAP models induced by L-arginine or sodium taurocholate, Tet may exert a protective effect on pancreatic tissue by inhibiting mechanisms such as NF - κ B and SOCE signaling pathways, but its specific molecular mechanism has not been elucidated [13, 16-18].
This study aims to replicate a mouse SAP model and explore the mechanism of Tet in treating SAP from the perspectives of calcium signaling, mitochondrial function, and ERS, providing experimental evidence for the clinical application of Tet.
Materials and Methods
1 animal
SPF grade male C57BL/6 mice, 6-8 weeks old, 20-25 g, purchased from Guangdong Medical Experimental Animal Center, license number: SCXK (Yue) 2022-0002. Throughout the experiment, the mice lived in a sterile, constant temperature, and ventilated cage, with free access to food and water. Passed the ethical review of the Experimental Animal Center of Jinan University.
2 main reagents
Tet（Abcam）； Taurolitocholic acid sodium salt (TLCS), palmitoleic acid (POA), amylase detection kit, myeloperoxidase (MPO) detection kit, and reactive oxygen species fluorescent probe DCFH-DA (Sigma Aldrich); Tribromoethanol and sodium tert pentanol (Aladdin); Malondialdehyde (MDA) detection kit, glutathione peroxidase (GSH Px) detection kit, superoxide dismutase (SOD) and catalase (CAT) detection kit (KeyGEN Biotech); Interleukin-6 (IL-6) ELISA kit (R&D Systems); SDS-PAGE rapid gel kit and I anti diluent (Beyotime); Chemiluminescent developer (Meso Scale Diagnostics); loading buffer（5×；Biosharp）； Protein pre staining marker and BCA protein detection kit (Thermo Fisher); Electrophoresis buffer, transfer buffer, TBS, and PBS (Servicebio); C/EBP homologous protein (CHOP) mouse antibody (Cat # 2895S; 1:1000; Cell Signaling Technology); Phosphorylated protein kinase R-like endoplasmic reticulum kinase (p-PERK); Thr980) monoclonal antibody (Cat # MA5-15033; 1:2000; Invitrogen); PERK polyclonal antibody (Cat # 24390-1-AP; 1:1000; Proteintech); Anti β - actin mouse antibody (Cat # PTM-5018; 1:1000; TM BIO); Anti glucose regulated protein 78 (GRP78) antibody (Cat # SC-13539; 1:1000; Santa Cruz Biotechnology).
3 main instruments
High speed tissue grinding instrument (Servicebio); Fully automatic inverted fluorescence microscope, fluorescence stereomicroscope, and confocal laser microscope (Leica); High speed low-temperature freeze centrifuge (Eppendorf); JB-3 type constant temperature magnetic stirrer (Jiangsu Jarier Electric Co., Ltd., China); Multi functional microplate imaging device (BioTek); AUIANE gel imaging system (UVITEC); Vertical electrophoresis instrument (Bio Rad).
4 main methods
4.1 Acute isolation of mouse PACs
PACs were prepared using acute separation method [19-20]. Anesthetize mice by intraperitoneal injection with tribromoethanol, and fix them in a supine position on the surgical pad. Disinfect the abdomen with alcohol cotton balls; Remove hair and cut open the abdominal cavity, take out the pancreas and place it in an EP tube, and digest it with 1 mL of type IV collagenase; Add an appropriate amount of NaHEPES solution to the EP tube, blow the pancreas several times with a pipette, let it stand for 10-15 seconds, collect the supernatant into a 15 mL centrifuge tube, repeat this step multiple times, and centrifuge the supernatant at 160.9 × g for 1 minute; Discard the supernatant, add 2 mL of NaHEPES solution, blow evenly, and centrifuge again at 160.9 × g for 1 minute; Discard the supernatant and resuspend the cells in NaHEPES solution to prepare a cell suspension.
4.2 Calcium ion fluorescence imaging
Fluo-4 fluorescent probe was incubated with PACs at a final concentration of 5 μ mol/L in the dark at room temperature for 30 minutes, centrifuged at 160.9 × g for 2 minutes, and the supernatant was discarded. NaHEPES was added to resuspend the cells to obtain PACs suspension [19-20]. Add corresponding injury drugs (500 μ mol/L TLCS or 50 μ mol/L POA) or therapeutic drugs+injury drugs (100 μ mol/L Tet+500 μ mol/L TLCS or 100 μ mol/L Tet+50 μ mol/L POA) according to grouping in the perfusion device. Confocal adjustment of excitation light wavelength of 488 nm and emission light wavelength of 510-560 nm. Place the cells in a matching perfusion channel, adhere for 5 minutes, and perform real-time fluorescence recording. After recording for 120 seconds, open the perfusion device and perfuse the corresponding medication, recording for 1600 seconds.
4.3 Propidium iodide (PI) fluorescence staining
Add NaHEPES, 100 μ mol/L Tet, 500 μ mol/L TLCS, 50 μ mol/L POA, 100 μ mol/L Tet+500 μ mol/L TLCS, and 100 μ mol/L Tet+50 μ mol/L POA into 6 EP tubes containing cell suspensions; Add 5 μ L of PI dye to each EP tube, blow and mix well; Add the cell fluid from the EP tube to the confocal microscopy dish and attach it to the wall; Confocal setting excitation wavelength of 535 nm, emission wavelength of 617 nm, 1024 × 1024 pixels, captured with a 63 times oil mirror.
4.4 Mitochondrial membrane potential detection
TMRM fluorescent probe was incubated with PACs at a final concentration of 20 μ mol/L in the dark at room temperature for 20 minutes. The cells were centrifuged at 160.9 × g for 2 minutes, and the supernatant was discarded. NaHEPES was added to resuspend the cells to obtain PACs suspension. Add corresponding injury drugs (500 μ mol/L TLCS or 50 μ mol/L POA) or therapeutic drugs+injury drugs (100 μ mol/L Tet+500 μ mol/L TLCS or 50 μ mol/L POA) according to grouping in the perfusion device. Confocal adjustment of excitation light wavelength of 549 nm and emission light wavelength of 573 nm. Place the cells in a matching perfusion channel, adhere to the wall for 5 minutes, and perform real-time fluorescence recording. After 120 seconds of recording, open the perfusion device and perfuse the corresponding medication for a total of 1200 seconds.
4.5 ROS detection
Incubate the cell suspension at room temperature with 10 μ mol/L live cell fluorescent probe DCFH-DA in the dark for 30 minutes, centrifuge at 160.9 × g for 2 minutes, discard the supernatant, and resuspend the cells with NaHEPES to obtain PACs suspension. Subsequently, the fluorescence was measured every 30 seconds using a BioTek CytationTM 5 multifunctional microplate imaging instrument for 2400 seconds, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.
4.6 Copying SAP mouse model
The SAP mouse model was replicated using the method described in the published literature [19-20]. 35 healthy male C57BL/6 mice (6-8 weeks old, 20-25 g) were randomly divided into 7 groups, with 5 mice in each group: (1) Control (Ctrl) group: mice were orally administered with physiological saline (10 mg/kg); (2) Tet group: mice were orally administered Tet (10 mg/kg) [18]; (3) Sham surgery group: After laparotomy and abdominal closure, physiological saline (10 mg/kg) was administered orally; (4) Alcoholic SAP (fatty acid ethyl ester induced AP, FAEE-AP) group: 200 μ L PBS+POA (150 mg/kg)+ethanol (ethyl alcohol, EtOH) was injected intraperitoneally on one side; 1.35 g/kg), with an interval of 1 hour, 200 μ L of PBS+POA (150 mg/kg)+EtOH (1.35 g/kg) were injected intraperitoneally on the other side [19-20]; (5) Bile SAP (TLCS induced AP, TLCS induced AP, TLCS-AP) group: After intraperitoneal injection of tribromoethanol (500 mg/kg) anesthesia, 50 μ L of TLCS (3 mmol/L) was infused into the pancreatic duct at a rate of 5 μ L/min using a microinjection pump [19-20]; (6) Tet treatment for FAEE-AP (Tet+FAEE-AP) group: Create a model according to the FAEE-AP group, and then administer Tet (10 mg/kg) orally; (7) Tet treatment for TLCS-AP (Tet+TLCS-AP) group: Create a model according to the TLCS-AP group, and administer Tet (10 mg/kg) by gavage after 1 hour. After 24 hours of the above treatment, the mice were anesthetized, eye blood was collected, and the lungs and pancreas of the mice were dissected.
4.7 Histopathological observation
After removing the pancreas, shave off excess adipose tissue and mesentery on the surface, rinse twice in PBS, and then absorb the water on filter paper. Soak and fix in formaldehyde solution for subsequent paraffin embedded sections. Observe the tissue structure under a 400x microscope and score the pancreas from three aspects: tissue edema, inflammatory infiltration, and PACs necrosis.
4.8 Biochemical index testing
After collecting blood from the eyeball, let it stand at room temperature for 1 hour, centrifuge at 4 ℃ and 1006.2 × g for 15 minutes, and then collect the supernatant. Determination of amylase by coupling enzyme colorimetric method; Enzyme linked immunosorbent assay was used to determine the inflammatory factor IL-6; Determination of MDA by thiobarbituric acid substrate method; Determination of SOD by nitroblue tetrazole colorimetric method; Enzyme catalyzed reaction determination of GSH Px; Visible light method for detecting CAT. Extract MPO from pancreatic tissue and lung tissue using 4-fold volume buffer, centrifuge at 13000 × g for 10 minutes to obtain the supernatant as the sample solution, and measure MPO using enzyme coupled colorimetric method.
4.9 Immunohistochemistry
Pancreatic tissue sections were routinely deparaffinized to water, repaired with citrate buffer at high temperature and high pressure, and incubated with 3% H2O2 at room temperature in the dark for 25 minutes to block endogenous peroxidase activation; Add 3% bovine serum albumin dropwise into the histological circle to evenly cover the tissue, and seal at room temperature for 30 minutes; Gently shake off the blocking solution, add CHOP antibody prepared in a certain ratio (1:1000) onto the slice, and incubate overnight at 4 ℃ in a wet box; After washing, add II antibody to cover the tissue and incubate at room temperature for 50 minutes; DAB staining, hematoxylin counterstaining of cell nuclei, followed by dehydration and microscopic examination.
4.10 Western blot
After cutting the pancreatic tissue into pieces, add an appropriate amount of protease lysis buffer and mix well on ice for homogenization; Centrifuge at 4 ℃ and 1006.2 × g for 15 minutes, and collect the supernatant; Quantify protein using BCA method, add loading buffer (5 ×) and lysis buffer as needed to each sample, and then boil and denature; SDS-PAGE protein separation, wet transfer to PVDF membrane, and block with 5% skim milk for 1 hour; Add the first antibody (GRP78, PERK, p-PERK, CHOP, and β - actin antibodies) and shake overnight at 4 ℃ to recover the first antibody. Incubate with the second antibody at room temperature for 1 hour and wash the membrane with TBST. Drop the developer, and take photos with the gel luminous imaging machine.
5 Statistical processing
Perform statistical analysis on all data using SPSS 13.0 and GraphPad Prism 8. The data is expressed as mean ± standard error (mean ± SEM). Perform one-way ANOVA for inter group comparison. The difference is statistically significant with P<0.05.

result
Tet inhibits calcium overload induced by POA or TLCS in mouse PACs and reduces the degree of PACs necrosis
After extracting PACs and placing them on the wall, slowly perfuse them using a perfusion device; Real time microscopic recording of Ca2+levels in live cells, as shown in Figure 1A. As shown in Figures 1B-E, POA (50 μ mol/L) or TLCS (500 μ mol/L) can induce an abnormal increase in Ca2+levels within PACs; After pre incubation with Tet (100 μ mol/L or 500 μ mol/L) for 10 minutes, POA or TLCS did not stimulate PACs to undergo calcium overload (P<0.01), but there was no significant difference in the effect of Tet at 100 μ mol/L and 500 μ mol/L (P>0.05). As shown in Figure 1F~I, POA (50 μ mol/L) or TLCS (500 μ mol/L) was first used to induce an increase in Ca2+levels in PACs, followed by treatment with Tet (100 μ mol/L). It can be seen that under the action of Tet, POA or TLCS induced a slow decrease in Ca2+levels in PACs (P<0.01). As shown in Figure 1J, compared with the Ctrl group, the POA group and TLCS group showed a significant increase in necrotic PACs (P<0.01); After treatment with Tet (100 μ mol/L), the proportion of necrotic PACs in the corresponding group significantly decreased (P<0.01).
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Figure 1: Tet inhibits POA or TLCS induced calcium overload and necrosis in PACs
Figure 1 Tetrandrine （Tet） inhibited palmitoleic acid （POA）- or taurolithocholic acid sodium salt （TLCS）-induced calcium overload and necrosis in PACs. A：transmitted （TM） light and Fluo-4 fluorescence images of the cell cluster （scale bar=10 μm）； B~E：POA （50 μmol/L） or TLCS （500 μmol/L）-evoked elevated ［Ca2+］i plateau was siginificantly reduced by pretreatment with high or low concentration of Tet， and C and E were the areas under the curve （AUC） of the traces in B and D， respectively （n=7~13）； F~I：average traces and the AUC showing the inhibitory effect of low concentration of Tet （100 μmol/L） on POA （50 μmol/L） or TLCS （500 μmol/L）-evoked elevated ［Ca2+］i plateau （n=7~13）； J：representative images of TM Light and propidium iodide （PI） staining cells （scale bar=10 μm）， showing that Tet （100 μmol/L） effectively protected against POA （50 μmol/L） or TLCS （500 μmol/L）-induced pancreatic acinar necrosis （n>250 cells/group）. Mean±SEM. △△P<0.01 vs Ctrl group； ** P<0.01 vs POA group； ## P<0.01 vs TLCS group.
2 Tet improves POA or TLCS induced mitochondrial membrane potential reduction in mouse PACs
As shown in Figure 2, first, POA (50 μ mol/L) or TLCS (500 μ mol/L) was used to induce mitochondrial membrane potential depolarization in PACs, and then Tet (100 μ mol/L) was administered to PACs for treatment. It was observed that Tet could alleviate the decrease in mitochondrial membrane potential induced by POA or TLCS in PACs (P<0.01).
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Figure 2: Tet alleviates the mitochondrial membrane potential decrease induced by POA or TLCS in PACs
Figure 2 Tetrandrine （Tet） alleviated palmitoleic acid （POA）- or taurolithocholic acid sodium salt （TLCS）-induced mitochondrial membrane potential decrease in PACs. A：TMRM fluorescence and transmitted （TM） light image of the cell cluster （scale bar=10 μm）； B~E：Tet （100 μmol/L） markedly reduced POA （50 μmol/L） or TLCS （500 μmol/L）-induced mitochondrial membrane potential decrease in PACs， and C and E were the areas under the curve （AUC） of the traces in B and D， respectively. Mean±SEM. n=6~10. **P<0.01 vs POA or TLCS group.

3 Tet reduces POA or TLCS induced ROS production in mouse PACs
As shown in Figures 3A, B, and D, POA (50 μ mol/L) or TLCS (500 μ mol/L) can induce excessive ROS production in PACs, and the use of Tet (100 μ mol/L) can significantly reduce the excessive accumulation of ROS caused by POA or TLCS in PACs (P<0.01). As shown in Figures 3C and D, Tet (100 μ mol/L) did not affect ROS production and showed no significant difference compared to the Ctrl group (P>0.05).
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Figure 3: Tet reduces POA or TLCS induced excessive ROS production in PACs
Figure 3 Tetrandrine （Tet） decreased palmitoleic acid （POA）- or taurolithocholic acid sodium salt （TLCS）-induced excessive ROS production in PACs. A：effect of Tet （100 μmol/L） on POA （50 μmol/L）-induced ROS production in PACs； B：effect of Tet （100 μmol/L） on TLCS （500 μmol/L）-induced ROS production in PACs； C：Tet （100 μmol/L） did not affect ROS production in PACs； D：quantitative analysis of the ROS production in A~C. Mean±SEM. n=6~10. **P<0.01 vs POA group； ## P<0.01 vs TLCS group.
The therapeutic effect of Tet on mouse SAP model
As shown in Figure 4A, no edema or inflammatory cell infiltration was observed in the Ctrl group and Tet group. The pancreatic lobules were tightly arranged, with regular cell arrangement and no obvious necrosis; Under light microscopy, the FAEE-AP and TLCS-AP groups showed significant edema, visible necrosis, infiltration of inflammatory cells, inflammatory exudation between lobules, and slight widening of leaf spaces; Compared with the control group, the Tet+FAEE-AP or Tet+TLCS-AP group had a denser and more complete pancreatic lobule structure, with only a small amount of inflammatory cell infiltration in the lobule area. The nuclei of PACs were clear, and there was no obvious swelling, degeneration, or vacuolar structure in the cytoplasm; Compared with the FAEE-AP group and TLCS-AP group, the pathological score of the Tet treatment group was significantly reduced (P<0.01). As shown in Figures 4B-E, serum amylase activity, serum IL-6 level, pancreatic MPO activity, and lung MPO activity were significantly increased in the FAEE-AP group and TLCS-AP group (P<0.01); After Tet treatment, all four indicators were significantly reduced (P<0.01).
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Figure 4: The effect of Tet on the pathological changes and inflammatory biochemical indicators of pancreatic tissue in mice with severe acute pancreatitis model
Figure 4 Effect of tetrandrine （Tet） on the histopathological changes and biochemical responses in the pancreas of severe acute pancreatitis （AP） mouse models. A：representative HE staining images of pancreatic tissue slides showed that Tet significantly improved the pathological scores in fatty acid ethyl ester （FAEE）-AP and taurolithocholic acid sodium salt （TLCS）-AP （scale bar=100 μm）； B~E：administration of Tet significantly reduced serum amylase， serum IL-6， pancreatic MPO activity and lung MPO activity in FAEE-AP and TLCS-AP mouse models. Mean±SEM. n=5. △△P<0.01 vs control （Ctrl） group； ** P<0.01 vs FAEE-AP group； ## P<0.01 vs TLCS-AP group.
The effect of Tet on pancreatic tissue ERS in SAP model mice
As shown in Figure 5A, only a small number of CHOP positive stained cells were present in the pancreatic tissues of the Ctrl and Tet groups, and the positive staining was mainly located in the cytoplasm, with no staining observed in the nucleus; However, in the FAEE-AP group and TLCS-AP group, the positive staining expression of CHOP was significantly enhanced compared to other groups, with the most significant expression in the inflammatory infiltration area and PACs necrosis area; After treatment with Tet, CHOP positive staining was significantly reduced (P<0.01). As shown in Figure 5B, compared with the FAEE-AP group or TLCS-AP group, the protein levels of GRP78, PERK, p-PERK, and CHOP were significantly reduced in the Tet treatment group (P<0.05).
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Figure 5: Tet alleviates endoplasmic reticulum stress in pancreatic tissue of mice with severe acute pancreatitis model
Figure 5 Tetrandrine （Tet） significantly reduced endoplasmic reticulum stress （ERS） in pancreatic tissue of severe acute pancreatitis （AP） mouse models. A：representative images of immunohistochemical staining showed that Tet significantly reduced the expression of CHOP in pancreatic tissues of severe AP mouse models ［scale bar=100 μm （upper row） or 30 μm （lower row）］； B：Western blot analysis of the expression levels of ERS-related proteins GRP78， PERK， p-PERK and CHOP in pancreatic tissues. Mean±SEM. n=5. *P<0.05， **P<0.01 vs fatty acid ethyl ester （FAEE）-AP group； # P<0.05， ##P<0.01 vs taurolithocholic acid sodium salt （TLCS）-AP group.
The effect of Tet on oxidative stress in SAP model mice
As shown in Figure 6, compared with the Ctrl group, the levels of serum peroxide MDA in the FAEE-AP group and TLCS-AP group were significantly increased (P<0.01), while the levels of serum antioxidants GSH Px, SOD, and CAT were significantly decreased (P<0.01); After treatment with Tet, serum MDA levels significantly decreased, while serum GSH Px, SOD, and CAT levels significantly increased (P<0.01).
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Figure 6: Tet inhibits oxidative stress in a mouse model of severe acute pancreatitis
Figure 6 Tetrandrine （Tet） markedly inhibited oxidative stress in mouse severe acute pancreatitis （AP） models. A：serum MDA content； B：serum GSH-Px activity； C：serum SOD activity； D：serum CAT activity. Mean±SEM. n=5. △△P<0.01 vs control （Ctrl） group； ** P<0.01 vs fatty acid ethyl ester （FAEE）-AP group； ## P<0.01 vs taurolithocholic acid sodium salt （TLCS）-AP group.

discuss
AP is one of the common digestive system emergencies, with approximately 20% of patients progressing to SAP. SAP is a severe clinical condition characterized by local inflammation of the pancreas leading to systemic multi organ dysfunction, characterized by rapid onset, rapid progression, multiple complications, and high mortality rate [21]. Due to the lack of specific and effective drug treatments for SAP, it is of great significance to search for effective targeted therapy strategies.
Calcium overload is a hallmark event in the onset of SAP, and pathological calcium signaling can promote the activation of enzymes and release of inflammatory mediators in PACs, induce PACs necrosis, and accelerate the progression of SAP [19-22]. This study first explored the concentration of Tet that inhibits calcium overload at the cellular level. Research has shown that pre incubation of acute isolated mouse PACs with Tet (100 μ mol/L or 500 μ mol/L) can effectively inhibit calcium overload induced by POA or TLCS, transforming it into calcium oscillations. In addition, treatment with Tet (100 μ mo/L) during the Ca2+response induced by POA or TLCS was observed to alleviate the degree of calcium overload. In our further experimental results, it was shown that Tet (100 μ mo/L) could also reduce the PACs necrosis rate induced by POA or TLCS to a level close to the control group. There is research confirming that calcium overload can cause endocytic vesicles in PACs to release active proteases into the cytoplasm and extracellular space, leading to cellular self digestion and damage to neighboring cells [23]. Therefore, Tet may intervene in the release of activated proteasomes (such as trypsin) into the intracellular and extracellular space by inhibiting calcium overload, thereby reducing the occurrence of PACs necrosis. In addition, Tet may act on pancreatic stellate cells or macrophages by regulating their calcium signaling, reducing the promoting effect on damaged PACs [24-26]. Previous studies have shown that the continuous increase in Ca2+concentration within PACs leads to mitochondrial calcium overload, resulting in depolarization of mitochondrial membrane potential and reduced ATP production [27]. The results of this experiment showed that Tet can prevent further decrease in mitochondrial membrane potential of PACs induced by POA or TLCS. ROS is a very important regulatory factor in the pathogenesis of SAP [28]. It can open the endoplasmic reticulum Ca2+channels IP3R and RyR, releasing a large amount of Ca2+. Elevated Ca2+in the cytoplasm can further disrupt endoplasmic reticulum protein folding, induce mitochondrial oxidative stress, lead to endoplasmic reticulum dysfunction, and promote ERS and unfolded protein response (UPR) [29]. Meanwhile, when excessive ROS accumulates in cells, it will lead to mitochondrial dysfunction [30]. Excessive oxidative stress and ERS are often accompanied by autophagic disorders, and persistent inflammatory responses and autophagic disorders can initiate cell death programs, exacerbating the pathogenesis of SAP. The experimental results showed that Tet can inhibit the excessive production of ROS in PACs induced by POA or TLCS. The above results suggest that Tet may intervene in PACs necrosis by inhibiting the occurrence of calcium overload, protecting mitochondrial function, and reducing oxidative stress damage.
In order to further verify the therapeutic effect of Tet on mouse SAP models, this study replicated two SAP mouse models by intraperitoneal injection of EtOH+POA and retrograde injection of TLCS into the bile duct. The results showed that the pancreatic tissue of SAP model mice exhibited edema accompanied by significant inflammatory infiltration and necrosis; The serum amylase activity, IL-6 level, and MPO activity in pancreatic and lung tissues were significantly increased, indicating successful modeling. After Tet treatment, the pathological damage scores of pancreatic and lung tissues, as well as various inflammatory biochemical indicators in serum, decreased. The endoplasmic reticulum is the primary calcium storage reservoir for PACs, and Ca2+released from acid stores such as zymogen granules and lysosomes may also have important implications for the initiation of acute pancreatitis [31-33]. Studies have shown that TPCs are the main ion channels mediating the release of Ca2+from acid stores [34], and TPC2 is the main type of TPCs within PACs [35]. The Ca2+release mediated by TPCs can further induce the promotion of Ca2+release in the endoplasmic reticulum, leading to ERS [36-37]. Various reasons can cause changes in the Ca2+homeostasis of the endoplasmic reticulum lumen, leading to calcium deprivation or calcium overload, which can induce the accumulation of unfolded or misfolded proteins on the endoplasmic reticulum, causing dysfunction of ER and ultimately leading to cell death [38-39]. We detected the positive expression of CHOP in pancreatic tissue of two types of SAP mice through immunohistochemistry, and observed that the positive staining rate of CHOP in pancreatic tissue of mice significantly increased after model replication. The positive staining cells were mainly concentrated in necrotic PACs and inflammatory cells. The GRP78-PERK-CHOP pathway is a key pathway for UPR response and can regulate endoplasmic reticulum homeostasis. Increased expression of GRP78 is one of the markers of ERS and UPR [40]; PERK is one of the important signaling pathways of ERS, which serves as a central regulator in UPR to regulate transcription. Continuous activation can induce upregulation of CHOP [41]. Further Western blot was used to detect the expression changes of the pancreatic tissue ERS signaling pathway GRP78/PERK/CHOP. It was found that the protein levels of GRP78, p-PERK, and CHOP in pancreatic tissue were significantly increased after model replication; The serum MDA level significantly increased, while the GSH Px, SOD, and CAT levels significantly decreased. However, the results of Tet treatment in the SAP group model showed that the positivity rate of CHOP in mouse pancreatic tissue was significantly reduced, and the protein expression levels of GRP78, p-PERK, and CHOP in pancreatic tissue were significantly downregulated; The serum MDA level significantly decreased, while the GSH Px, SOD, and CAT levels significantly increased. The results of this experiment suggest that the activation of the pancreatic tissue ERS signaling pathway GRP78/PERK/CHOP and changes in oxidative stress factor levels are involved in the progression of SAP, and their mechanisms may be related to calcium overload in PACs. Tet may inhibit the calcium release process induced by TPC2, thereby reducing the calcium influx mediated by the calcium pool manipulation channel Orai [42], ultimately inhibiting PACs calcium overload, reducing ERS and oxidative stress damage, and exerting a preventive and therapeutic effect on SAP.
In summary, the traditional Chinese medicine monomer Tet may inhibit calcium overload induced by POA or TLCS in mouse PACs by blocking TPCs, thereby reducing ERS and oxidative stress damage in mouse pancreatic tissue, improving mitochondrial function, and achieving the effects of protecting PACs and preventing SAP. This study can provide experimental evidence for the clinical application of Tet.

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Tetrandrine attenuates severe acute pancreatitis in mouse models by inhibiting calcium overload

FU Guodan ¹ZHU Fengfeng ²BAI Yuhui ³XU Liting ¹YU Lei ¹DUAN Ruibin ⁴AN Ting ⁴LÜ Changsheng ⁵SHI Liping ⁶ PENG Shuang ^5,7

（1. Department of Pathophysiology， School of Medicine， Jinan University， Key Laboratory of Pathophysiology， National Administration of Traditional Chinese Medicine， Guangzhou 510632， China； 2. Department of Hepatobiliary and Pancreas Surgery， The First Affiliated Hospital， University of South China， Hengyang 421001， China； 3. Graduate School， Guangzhou Sport University， Guangzhou 510500， China； 4. Department of Clinical Medicine， School of Medicine， Jinan University， Guangzhou 510632， China； 5. School of Sport and Health Sciences， Guangzhou Sport University， Guangzhou 510500， China； 6. Department of Urology， The First Affiliated Hospital of Jinan University， Guangzhou 510632， China； 7. Key Laboratory of Sports Technique， Tactics and Physical Function of General Administration of Sport of China， Scientific Research Center， Guangzhou Sport University， Guangzhou 510500， China ）

Abstract:

AIMTo investigate the therapeutic effect of tetrandrine （Tet） on mouse in vivo and in vitro models of severe acute pancreatitis （SAP） and its mechanisms.

METHODS（1） The pancreatic tissues of C57BL/6 mice were freshly isolated to obtain pancreatic acinar cells （PACs）. The cells were randomly divided into blank control （Ctrl） group， Tet group， injury ［induced by palmitoleic acid （POA） or taurolithocholic acid sodium salt （TLCS）］ groups and Tet treatment （Tet+POA or Tet+TLCS） groups. The Fluo-4 fluorescent probe was used to measure cytoplasmic calcium concentration. Propidium iodide staining was used to detect necrotic cells. The TMRM fluorescent probe was used to measure the changes of mitochondrial membrane potential. Reactive oxygen species （ROS） was detected by DCFH-DA fluorescent probe. （2） Thirty-five 8-week-old healthy male C57BL/6 mice were randomly selected and divided into Ctrl group， sham group， Tet group， fatty acid ethyl ester-induced acute pancreatitis （FAEE-AP） group， TLCS-induced acute pancreatitis （TLCS-AP） group， Tet+FAEE-AP group and Tet+TLCS-AP group， with 5 mice in each group. The morphological changes of the pancreas were observed using HE staining. The positive staining rates of C/EBP homologous protein （CHOP） in the pancreas were observed by immunohistochemistry. The expression of glucose-regulated protein 78 （GRP78）， protein kinase R-like endoplasmic reticulum kinase （PERK）， p-PERK and CHOP was detected by Western blot. Serum levels of amylase， interleukin-6 （IL-6）， malondialdehyde （MDA）， glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD） and catalase （CAT） were assayed. Myeloperoxidase （MPO） activity was detected in mouse pancreatic and lung tissues.

RESULTSTreatment with Tet effectively inhibited POA- or TLCS-induced calcium overload and necrosis in PACs， attenuated the decrease in mitochondrial membrane potential， and reduced ROS production in vitro （P<0.01）. Compared with Ctrl group， the pancreatic pathological score， serum amylase activity， serum IL-6 level， and MPO activity in the pancreas and lung in vivo significantly increased in SAP groups （P<0.01）. However， the administration of Tet significantly alleviated the tissue damage and inflammatory response （P<0.01）. Compared with SAP groups， Tet reduced the expression of endoplasmic reticulum stress-related GRP78/PERK/CHOP signaling pathway （P<0.01）， decreased serum peroxide MDA， and increased antioxidants GSH-Px， SOD and CAT （P<0.01）.

CONCLUSIONTreatment with Tet effectively attenuates SAP in mouse models by the inhibition of calcium overload， the alleviation of mitochondrial membrane potential decrease， and the reduction of endoplasmic reticulum stress response and oxidative stress damage to protect against the necrosis of PACs.

Keywords: tetrandrine；calcium overload；endoplasmic reticulum stress；oxidative stress；severe acute pancreatitis