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. 2021 Oct;10(10):3030-3044.
doi: 10.21037/gs-21-655.

Protective effect of Dachengqi decoction on the pancreatic microcirculatory system in severe acute pancreatitis by down-regulating HMGB-TLR-4-IL-23-IL-17A mediated neutrophil activation by targeting SIRT1

Jia Wang #  1 , Yang Zou #  2 , Dan Chang  2 , Da-Qing Hong  2 , Jiong Zhang  2
Affiliations

Protective effect of Dachengqi decoction on the pancreatic microcirculatory system in severe acute pancreatitis by down-regulating HMGB-TLR-4-IL-23-IL-17A mediated neutrophil activation by targeting SIRT1

Jia Wang et al. Gland Surg. 2021 Oct.

Abstract

Background: Dachengqi decoction (DCQD), one of classic prescription of Chinese herbal medicine has been widely used in clinic to treat severe acute pancreatitis (SAP). The damage of pancreatic microcirculation plays key pathogenesis of SAP. However, little is known about the molecular pharmacological activity of DCQD on pancreatic microcirculation in SAP.

Methods: Sodium taurodeoxycholate and cerulein were used to establish model of SAP in vitro and in vivo, respectively. The pancreatic pathological morphology, wet weight ratio, myeloperoxidase (MPO) activity, cell viability and microcirculatory function of the pancreas, as well as serum lipase and amylase expressions were evaluated. The expression levels of SIRT1, acety-HMGB1, TLR-4, HMGB1, IL-23, IL-17A, neutrophil chemokines (KC, LIX, and MIP-2), and inflammation-related factors (IL-6, IL-1β, and TNF-α), the translocation of HMGB1 and the interaction of SIRT-HMGB1 in the pancreas and serum were determined by ELISA real-time PCR, western blotting and immunoprecipitation.

Results: In vivo studies showed that DCQD or neutralizing antibody (anti-23p19 or anti-IL-17A) could all significantly decrease lipase, amylase activity, down-regulate the expression of CD68, Myeloperoxidase (MPO), wet/weight, IL-1β, IL-6, TNF-α, and neutrophil chemokines (KC, LIX, MIP-2), alleviate pathological injury and improve pancreatic microcirculatory function in rats with SAP. Furthermore, DCQD remarkably increased SIRT1 expression, promoted SIRT1 and HMGB1 combination, reduced HMGB1 translocation from nuclear to cytoplasm, and alleviated the expression of acetyl-HMGB1, HMGB1, IL-17A, TLR-4, and IL-23 in vitro and in vivo with SAP. However, the intervention with EX527 (SIRT1 inhibitor) or r-HMGB1 (recombinant HMGB1) obliviously reverses the above mentioned influence mentioned above of DCQD in SAP. In vitro, we confirmed that DCQD could decrease HMGB1 acetylation, migration, and release, and improve the decline of cell viability, SIRT1 expression and SIRI-HMGB1 combination induced by cerulean with promoting macrophage to release IL-23 by relying on the HMGB1/TLR-4 way.

Conclusions: DCQD treatment improves SAP-induced pancreatic microcirculatory dysfunction by inhibiting neutrophil-mediated inflammation via inactivating HMGB1-TLR-4-IL-23-IL-17A signaling by targeting SIRT1.

Keywords: Dachengqi decoction (DCQD); HMGB1-TLR-4-IL-23-IL-17A; Severe acute pancreas (SAP); neutrophil; pancreatic microcirculatory.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/gs-21-655). Dr. JW reported that the study was funded by the Foundation of Sichuan Provincial People’s Hospital (No. 2017LY11). Dr. JZ reported that the study was funded by the Foundation of Sichuan Provincial People’s Hospital (No. 2020LY07) and scientific research project of Sichuan Provincial Department of Science and Technology (No. 2020YJ0179). The other authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The effect of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on wet/weight, amylase and lipase activity, surviving rats, and AR42J cell viability in SAP. (A) The influence of DCQD, EX527 and r-HMGB1 on wet/weight, amylase and lipase activity. (B) The influence of anti-IL-17A and anti-IL-23p19 on wet/weight, amylase and lipase activity. (C) The influence of DCQD, EX527, anti-IL-17A, r-HMGB1 and anti-IL-23p19 on the survive rats in SAP. (D) The effect of DCQD, EX527 and anti-IL-17A, r-HMGB1and anti-IL-23p19 on the AR42J cell viability in SAP. (E) The effect of anti-IL-17A and anti-IL-23p19 on the AR42J cell viability in SAP; ***, P<0.001 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A vs. SAP); ***, P<0.001 (SAP vs. Control). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Figure 2
Figure 2
The influence of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on pancreatic histopathological morphology, and CD68 and MPO expression in SAP. (A) Representative results from HE stains (magnification ×200) on the histopathological morphology of the pancreas and immunohistochemistry for CD68 and MPO; (B) the damage score was evaluated by semiquantitative analysis. (C) CD68 and MPO expression were evaluated by semiquantitative analysis in SAP. ***, P<0.001 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); @, P<0.05 (anti-IL-23p19 vs. SAP); $, P<0.05 (anti-IL-23p19 vs. SAP). The blue arrows indicate the positive place. DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Figure 3
Figure 3
The effect of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on pancreatic microcirculatory function in SAP. (A) The effect of DCQD, EX527, and r-HMGB1 on blood flow velocity, number of functional blood vessel, RBC flow and number of blood vessel. (B) The influence of anti-IL-17A and anti-IL-23p19 on the blood flow velocity, number of functional blood vessel, RBC flow and number of blood vessel. ***, P<0.001 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A vs. SAP). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Figure 4
Figure 4
The influence of DCQD, EX527, anti-IL-17A, r-HMGB1, and anti-IL-23p19 on the level of IL-23, HMGB1, IL-1β, IL-17A, TNF-α, and IL-6 in SAP. (A) ELISA was used to assess the influence of DCQD, EX527 and r-HMGB1 on the serum level of IL-23, HMGB1, IL-1β, IL-17A, TNF-α and IL-6 in SAP; (B) RT-PCR was used to assess the effect of DCQD, EX527 and r-HMGB1 on the pancreatic mRNA of IL-23, HMGB1, IL-1β, IL-17A, TNF-α and IL-6 in SAP; (C) ELISA was used to examine the effect of DCQD, EX527, and r-HMGB1 on the pancreatic level of IL-23, HMGB1, IL-1β, IL-17A, TNF-α and IL-6 in SAP; (D) the ELISA was used to test the influence of anti-IL-17A and anti-IL-23p19 on the serum level of TNF-α, IL-6 and IL-1β in SAP; (E) ELISA was used to measure the influence of anti-IL-17A and anti-IL-23p19 on the pancreatic level of TNF-α, IL-6 and IL-1β in SAP; (F) RT-PCR was used to measure the influence of anti-IL-17A and anti-IL-23p19 on pancreatic mRNA of TNF-α, IL-6 and IL-1β in SAP; (G) ELISA and RT-PCR were used to evaluate the influence of anti-IL-23p19 on the level of IL-17A in SAP. *, P<0.05 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A vs. SAP). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Figure 5
Figure 5
The influence of DCQD, EX527, anti-IL-17A, r-HMGB1 and anti-IL-23p19 on MPO activity and expression of MIP-2, KC, and LIX in SAP. (A) ELISA was exploited to evaluate the influence of DCQD, r-HMGB1, and EX527 on MPO activity in SAP; (B) ELISA was used to assess the influence of anti-IL-17A and anti-IL-23p19 on MPO activity in SAP; (C) ELISA was used to analyze the influence of DCQD, r-HMGB1, and EX527 on serum MIP-2, KC, and LIX level in SAP; (D) ELISA was exploited to evaluate the influence of DCQD, r-HMGB1, and EX527 on MIP-2, KC and LIX expression in SAP; (E) RT-PCR was exploited to measure the influence of DCQD, r-HMGB1 and EX527 on the MIP-2, KC and LIX mRNA expression in SAP; (F) ELISA was designated to measure the influence of anti-IL-17A and anti-IL-23p19 on the MIP-2, KC and LIX serum expression in SAP; (G) ELISA was designated to evaluate the influence of anti-IL-17A and anti-IL-23p19 on the MIP-2, KC and LIX pancreatic expression in SAP; (H) RT-PCR was used to evaluate the influence of anti-IL-17A and anti-IL-23p19 on the MIP-2, KC, and LIX pancreatic mRNA in SAP; ***, P<0.001 (SAP vs. Sham); ***, P<0.001 (SAP vs. Control); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD); &, P<0.05 (r-HMGB1 vs. DCQD); ^, P<0.05 (anti-IL-23p19 vs. SAP); #, P<0.05 (anti-IL-17A vs. SAP). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis; MPO, myeloperoxidase.
Figure 6
Figure 6
The influence of DCQD on the SIRT1, acety-HMGB1 and TLR-4 expression, and HMGB1 translocation, SIRT1 activity, and SIRT1-HMGB1combination in the pancreas in the SAP. Western blotting, immunoprecipitation, and ELISA were designated to evaluate SIRT1, acety-HMGB1 and TLR-4 expression, HMGB1 translocation, SIRT1 activity, and SIRT1-HMGB1combination in vitro and in vivo. (A) Immunoprecipitation was exploited to assay the combination of SIRT1 and HMGB1 in the pancreas; (B) immunoprecipitation was exploited to assay the combination of SIRT1 and HMGB1 in the AR42J cell; (C) the SIRT1 activity in the pancreas; (D) SIRT1 activity in AR42Jcell; (E) typical result for Western blotting analysis SIRT1 in the pancreases; (F) rat SIRT1, Acety-HMGB1, HMGB1 in nuclear and cytoplasm, and TLR-4 expression were evaluated by semiquantitative analysis; (G) typical result for the Western blot analysis SIRT1, Acety-HMGB1, HMGB1 in nuclear and cytoplasm, and TLR-4 in the cell; (H) the expression of AR42J cell SIRT1 was evaluated by semiquantitative analysis: (I) typical results for Western blotting on TLR-4 in SAP in rats; (J) rat TLR-4 expression was assessed by semiquantitative analysis; (K) typical results for Western blot on TLR-4 in the AR42J cell; (L) cell TLR-4 expression was assessed by semi-quantitative analysis. ***, P<0.001 (SAP vs. Sham); #, P<0.05 (SAP vs. DCQD); ^, P<0.05 (EX527 vs. DCQD). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Figure 7
Figure 7
The influence of DCQD on the expression of TLR-4 and IL-23 in SAP. (A) The influence of DCQD on the level of TLR-4 and IL-23 mRNA in RAW264.7 was detected through RT-PCR; (B) ELISA was designated to evaluate the influence of r-HMGB1 on IL-23 expression in the supernatant in SAP. ***, P<0.001 (SAP vs. Control); #, P<0.001, (DCQD vs. SAP); ^, P<0.05 (DCQD vs. siRNA-TLR-4); &, P<0.05 (DCQD vs. plasmid-TLR-4). DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
Figure 8
Figure 8
The signal pathway of DCQD on pancreatic microcirculatory in SAP. DCQD, Dachengqi decoction; SAP, severe acute pancreatitis.
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