Therapeutic Effect of Combining Anisodamine With Neostigmine on Local Scar Formation Following Roux-en-Y Choledochojejunostomy in a Novel Rat Model

doi:10.3389/fphar.2021.700050

. 2021 Sep 29:12:700050.

doi: 10.3389/fphar.2021.700050. eCollection 2021.

Therapeutic Effect of Combining Anisodamine With Neostigmine on Local Scar Formation Following Roux-en-Y Choledochojejunostomy in a Novel Rat Model

Shao-Cheng Lyu¹, Jing Wang¹, Wen-Li Xu¹, Han-Xuan Wang¹, Fei Pan¹, Tao Jiang¹, Qiang He¹, Ren Lang¹

Affiliations

PMID: 34658849
PMCID: PMC8511430
DOI: 10.3389/fphar.2021.700050

Therapeutic Effect of Combining Anisodamine With Neostigmine on Local Scar Formation Following Roux-en-Y Choledochojejunostomy in a Novel Rat Model

Shao-Cheng Lyu et al. Front Pharmacol. 2021.

. 2021 Sep 29:12:700050.

doi: 10.3389/fphar.2021.700050. eCollection 2021.

Authors

Shao-Cheng Lyu¹, Jing Wang¹, Wen-Li Xu¹, Han-Xuan Wang¹, Fei Pan¹, Tao Jiang¹, Qiang He¹, Ren Lang¹

Affiliation

¹ Department of Hepatobiliary and Pancreaticosplenic Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing, China.

PMID: 34658849
PMCID: PMC8511430
DOI: 10.3389/fphar.2021.700050

Abstract

Background: The present study aimed to explore the potential effect of combining anisodamine with neostigmine on local scar formation following Roux-en-Y choledochojejunostomy (RCJS) in a novel rat model. Methods: The biliary obstruction model of Sprague Dawley (SD) rats was established in advance, and 54 rats were divided into nine groups randomly (sham operation group, anisodamine group, neostigmine group, combination group, and control group). Anisodamine (25 mg/kg) and neostigmine (50 μg/kg) were injected to the abdominal cavity separately or simultaneously for 1 week since the first day after surgery according to their allocated intervention, while the same amount of saline (0.5 ml) was injected intraperitoneally in the control group. Indexes including body weight, the diameter of the common bile duct, liver function, inflammatory indexes, and the condition of scar formation in different groups at certain time were evaluated in our study. Results: Recovery of liver function (ALT, AST, TB, DB, and GGT) and systematic inflammation indexes (CRP, TNF-α, and IL-1β) in the combination group was prior to that in the control group (p < 0.05), while no statistical difference in the serum level of IL-10 was observed among groups. Rats in the combination group represented a wider anastomotic diameter and lower expression of α-SMA and TGF-β1 at anastomotic stoma compared to the control group (p < 0.05). Histopathological staining showed slighter proliferation of collagen and smooth muscle fibers in rats' bile duct wall and less local scar formation at anastomotic stoma compared to the control group. Conclusion: The combination of anisodamine and neostigmine can alleviate local and systemic inflammatory response, promote the recovery of liver function, and reduce scar formation in rats after the RCJS procedure.

Keywords: Roux-en-Y choledochojejunostomy; anisodamine; inflammatory response; neostigmine; scar formation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

**FIGURE 1**
Changes of weight and anastomotic diameter in rats at different time points after the RCJS procedure (* refers to the comparison between the two groups, p < 0.05). Comparison of **(A)** weight (g) and **(B)** anastomotic diameter (mm) of rats in different groups at different time points.

**FIGURE 2**
Changes of liver function in rats at different time points after the RCJS procedure (* refers to the comparison between the two groups, p < 0.05). Comparison of **(A)** ALT (U/L), **(B)** AST (U/L), **(C)** TB (µmol/L), **(D)** DB (µmol/L), and **(E)** GGT (U/L) at different time points in rats of each group.

**FIGURE 3**
Changes of inflammatory indexes in rats at different time points after the RCJS procedure (* refers to the comparison between the two groups, p < 0.05). Comparison of **(A)** CRP (ng/ml), **(B)** TNF-α (pg/ml), **(C)** IL-1β (pg/ml), and **(D)** IL-10 (pg/ml) at different time points in rats of each group.

**FIGURE 4**
HE staining of anastomotic stoma in different groups after the RCJS procedure (×200). **(A)** Sham operation group. **(B)** Control group at 1 week after the RCJS procedure. **(C)** Control group at 1 month after the RCJS procedure. **(D)** Anisodamine intervention group at 1 week after the RCJS procedure. **(E)** Anisodamine intervention group at 1 month after the RCJS procedure. **(F)** Neostigmine intervention group at 1 week after the RCJS procedure. **(G)** Neostigmine intervention group at 1 month after the RCJS procedure. **(H)** Combination group at 1 week after the RCJS procedure. **(I)** Combination group at 1 month after the RCJS procedure.

**FIGURE 5**
Masson’s staining of anastomotic stoma in different groups after the RCJS procedure (×200). **(A)** Sham operation group. **(B)** Control group at 1 week after the RCJS procedure. **(C)** Control group at 1 month after the RCJS procedure. **(D)** Anisodamine intervention group at 1 week after the RCJS procedure. **(E)** Anisodamine intervention group at 1 month after the RCJS procedure. **(F)** Neostigmine intervention group at 1 week after the RCJS procedure. **(G)** Neostigmine intervention group at 1 month after the RCJS procedure. **(H)** Combination group at 1 week after the RCJS procedure. **(I)** Combination group at 1 month after the RCJS procedure.

**FIGURE 6**
Immunohistochemical staining of α-SMA in the anastomotic stoma after RCJS in different groups (×200). **(A)** IHC staining of α-SMA in the sham operation group. **(B)** IHC staining of α-SMA in the control group at 1 week after the RCJS procedure. **(C)** IHC staining of α-SMA in the control group at 1 month after the RCJS procedure. **(D)** IHC staining of α-SMA in the anisodamine intervention group at 1 week after the RCJS procedure. **(E)** IHC staining of α-SMA in the anisodamine intervention group at 1 month after the RCJS procedure. **(F)** IHC staining of α-SMA in the neostigmine intervention group at 1 week after the RCJS procedure. **(G)** IHC staining of α-SMA in the neostigmine intervention group at 1 month after the RCJS procedure. **(H)** IHC staining of α-SMA in the combination group at 1 week after the RCJS procedure. **(I)** IHC staining of α-SMA in the combination group at 1 month after the RCJS procedure.

**FIGURE 7**
Immunohistochemical staining of TGF-β1 in the anastomotic stoma after RCJS in different groups (×200). **(A)** IHC staining of TGF-β1 in the sham operation group. **(B)** IHC staining of TGF-β1 in the control group at 1 week after the RCJS procedure. **(C)** IHC staining of TGF-β1 in the control group at 1 month after the RCJS procedure. **(D)** IHC staining of TGF-β1 in the anisodamine intervention group at 1 week after the RCJS procedure. **(E)** IHC staining of TGF-β1 in the anisodamine intervention group at 1 month after the RCJS procedure. **(F)** IHC staining of TGF-β1 in the neostigmine intervention group at 1 week after the RCJS procedure. **(G)** IHC staining of TGF-β1 in the neostigmine intervention group at 1 month after the RCJS procedure. **(H)** IHC staining of TGF-β1 in the combination group at 1 week after the RCJS procedure. **(I)** IHC staining of TGF-β1 in the combination group at 1 month after the RCJS procedure.

**FIGURE 8**
Comparison of the pathological staining MOD of anastomotic stoma in rats at different time points after the RCJS procedure (* refers to the comparison between the two groups, p < 0.05). Comparison of MOD values of **(A)** Masson’s staining, **(B)** α-SMA immunohistochemical staining, and **(C)** TGF-β1 immunohistochemical staining at different time points in rats of each group.

**FIGURE 9**
Changes of the expression levels of α-SMA and TGF-β1 in rats at different time points after the RCJS procedure (* refers to the comparison between the two groups, p < 0.05). Comparison of **(A)** α-SMA gene expression and **(B)** TGF-β1 gene expression among different groups of rats at different time points.

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References

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[1] Asano T., Natsume S., Senda Y., Sano T., Matsuo K., Kodera Y., et al. (2016). Incidence and Risk Factors for Anastomotic Stenosis of Continuous Hepaticojejunostomy After Pancreaticoduodenectomy. J. Hepatobiliary Pancreat. Sci. 23 (10), 628–635. 10.1002/jhbp.385 - DOI - PubMed

[2] Asano T., Natsume S., Senda Y., Sano T., Matsuo K., Kodera Y., et al. (2016). Incidence and Risk Factors for Anastomotic Stenosis of Continuous Hepaticojejunostomy After Pancreaticoduodenectomy. J. Hepatobiliary Pancreat. Sci. 23 (10), 628–635. 10.1002/jhbp.385 - DOI - PubMed

[3] Bettschart V., Clayton R. A. E., Parks R. W., Garden O. J., Bellamy C. O. C. (2002). Cholangiocarcinoma Arising After Biliary-Enteric Drainage Procedures for Benign Disease. Gut 51 (1), 128–129. 10.1136/gut.51.1.128 - DOI - PMC - PubMed

[4] Bettschart V., Clayton R. A. E., Parks R. W., Garden O. J., Bellamy C. O. C. (2002). Cholangiocarcinoma Arising After Biliary-Enteric Drainage Procedures for Benign Disease. Gut 51 (1), 128–129. 10.1136/gut.51.1.128 - DOI - PMC - PubMed

[5] Birgin E., Téoule P., Galata C., Rahbari N. N., Reissfelder C. (2020). Cholangitis Following Biliary-Enteric Anastomosis: A Systematic Review and Meta-Analysis. Pancreatology 20 (4), 736–745. 10.1016/j.pan.2020.04.017 - DOI - PubMed

[6] Birgin E., Téoule P., Galata C., Rahbari N. N., Reissfelder C. (2020). Cholangitis Following Biliary-Enteric Anastomosis: A Systematic Review and Meta-Analysis. Pancreatology 20 (4), 736–745. 10.1016/j.pan.2020.04.017 - DOI - PubMed

[7] Block L., Gosain A., King T. W. (2015). Emerging Therapies for Scar Prevention. Adv. Wound Care 4 (10), 607–614. 10.1089/wound.2015.0646 - DOI - PMC - PubMed

[8] Block L., Gosain A., King T. W. (2015). Emerging Therapies for Scar Prevention. Adv. Wound Care 4 (10), 607–614. 10.1089/wound.2015.0646 - DOI - PMC - PubMed

[9] Booij K. A. C., Coelen R. J., de Reuver P. R., Besselink M. G., van Delden O. M., Rauws E. A., et al. (2018). Long-term Follow-Up and Risk Factors for Strictures after Hepaticojejunostomy for Bile Duct Injury: An Analysis of Surgical and Percutaneous Treatment in a Tertiary center. Surgery 163 (5), 1121–1127. 10.1016/j.surg.2018.01.003 - DOI - PubMed

[10] Booij K. A. C., Coelen R. J., de Reuver P. R., Besselink M. G., van Delden O. M., Rauws E. A., et al. (2018). Long-term Follow-Up and Risk Factors for Strictures after Hepaticojejunostomy for Bile Duct Injury: An Analysis of Surgical and Percutaneous Treatment in a Tertiary center. Surgery 163 (5), 1121–1127. 10.1016/j.surg.2018.01.003 - DOI - PubMed

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Therapeutic Effect of Combining Anisodamine With Neostigmine on Local Scar Formation Following Roux-en-Y Choledochojejunostomy in a Novel Rat Model

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Therapeutic Effect of Combining Anisodamine With Neostigmine on Local Scar Formation Following Roux-en-Y Choledochojejunostomy in a Novel Rat Model

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Abstract

Conflict of interest statement

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