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. 2021 May 4:14:1201-1214.
doi: 10.2147/JPR.S303595. eCollection 2021.

Ulinastatin Exhibits Antinociception in Rat Models of Acute Somatic and Visceral Pain Through Inhibiting the Local and Central Inflammation

Mei-Xiang Zhan #  1   2 , Li Tang #  3 , Yun-Fei Lu #  4 , Huang-Hui Wu #  1 , Zhi-Bin Guo  1 , Zhong-Mou Shi  1 , Chen-Long Yang  1 , Yi-Qing Zou #  1   2 , Fei Yang  1   2   5 , Guo-Zhong Chen  1   2
Affiliations

Ulinastatin Exhibits Antinociception in Rat Models of Acute Somatic and Visceral Pain Through Inhibiting the Local and Central Inflammation

Mei-Xiang Zhan et al. J Pain Res. .

Abstract

Introduction: Ulinastatin, a broad-spectrum serine protease inhibitor, has been widely used to treat various diseases clinically. However, so far, the antinociceptive effect of ulinastatin remains less studied experimentally and the underlying mechanisms of ulinastatin for pain relief remain unclear. This study aimed to find evidence of the analgesic effect of ulinastatin on acute somatic and visceral pain.

Methods: The analgesic effect of ulinastatin on acute somatic and visceral pain was evaluated by using formalin and acetic acid-induced writhing test. The analgesic mechanism of ulinastatin was verified by detecting the peripheral inflammatory cell infiltration and spinal glial activation with hematoxylin-eosin (H&E) and immunohistochemistry staining.

Results: We found that both of intraperitoneal (i.p.) pre-administration and post-administration of ulinastatin could reduce the total number of flinching and the licking duration following intraplantar formalin injection in a dose-related manner. However, the inhibitory effect of ulinastatin existed only in the second phase (Phase 2) of formalin-induced spontaneous pain response, with no effect in the first phase (Phase 1). The formalin-induced edema and ulcer were also improved by i.p. administration of ulinastatin. Moreover, i.p. administration of ulinastatin was also able to delay the occurrence of acetic acid-induced writhing and reduced the total number of writhes dose-dependently. We further demonstrated that ulinastatin significantly decreased the local inflammatory cell infiltration in injured paw and peritoneum tissue under formalin and acetic acid test separately. The microglial and astrocytic activation in the spinal dorsal horn induced by intraplantar formalin and i.p. acetic acid injection were also dramatically inhibited by i.p. administration of ulinastatin.

Conclusion: Our results for the first time provided a new line of evidence showing that ulinastatin could attenuate acute somatic and visceral pain by inhibiting the peripheral and spinal inflammatory reaction.

Keywords: Ulinastatin; antinociception; formalin; inflammation; somatic pain; visceral pain.

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Conflict of interest statement

The authors have declared that no competing interests exist in this work.

Figures

Figure 1
Figure 1
Experimental design and timeline. (A) Schematic diagram for exploring the effect of UTI on formalin-induced somatic pain. (B) Schematic diagram for exploring the effect of UTI on acetic acid-induced visceral pain. i.p. intraperitoneal; i.pl. intraplantar; UTI, ulinastatin.
Figure 2
Figure 2
Effects of i.p. ulinastatin pre-treatment on formalin-induced spontaneous licking and flinching. (A) Curve graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the licking time at each 5 min interval during 60 min immediately after intraplantar formalin injection. (B) Column graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the mean total licking time in 60 min course immediately after intraplantar formalin injection. (C) Curve graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the spontaneous flinches at each 5 min interval during 60 min immediately after intraplantar formalin injection. (D) Column graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the mean total flinches in 60 min course after immediately intraplantar formalin injection. n=8/group, #P < 0.05 vs Saline, &P < 0.05 vs UTI 10,000 U/kg, two-way ANOVA followed by Tukey’s post hoc test.
Figure 3
Figure 3
Effects of i.p. ulinastatin post-treatment on formalin-induced spontaneous licking and flinching. (A) Curve graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the licking time at each 5 min interval during 60 min immediately after intraplantar formalin injection. (B) Column graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the mean total licking time in 60 min course immediately after intraplantar formalin injection. (C) Curve graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the spontaneous flinches at each 5 min interval during 60 min immediately after intraplantar formalin injection. (D) Column graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the mean total flinches in 60 min course after immediately intraplantar formalin injection. n=8/group, #P < 0.05 vs Saline, two-way ANOVA followed by Tukey’s post hoc test.
Figure 4
Figure 4
Effects of i.p. ulinastatin administration on acetic acid-induced spontaneous writhe. (A) Column graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the writhes latency from i.p. acetic acid injection. (B) Column graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the mean total writhes in 60 min course immediately after i.p. acetic acid injection. n=8/group, #P < 0.05 vs Saline, &P < 0.05 vs UTI 10,000 U/kg, one-way ANOVA followed by Tukey’s post hoc test. (C) Column graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the writhes latency from i.p. acetic acid injection. (D) Column graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the mean total writhes in 60 min course immediately after i.p. acetic acid injection. n=8/group, #P < 0.05 vs Saline, Student’s unpaired t-test.
Figure 5
Figure 5
Effects of i.p. ulinastatin administration on formalin-induced paw edema. (A) Curve graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the paw edema at 2 h and day 1, 3, 5, 7, 10 after intraplantar formalin injection. (B) Area under the curve (AUC) for different pre-treatment groups was calculated for statistical analysis. n=8/group, #P < 0.05 vs Saline, one-way ANOVA followed by Tukey’s post hoc test. (C) Curve graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the paw edema at 2 h and day 1, 3, 5, 7, 10 after intraplantar formalin injection. (D) Area under the curve (AUC) for post-treatment groups was calculated for statistical analysis. n=8/group, Student’s unpaired t-test.
Figure 6
Figure 6
Effects of i.p. ulinastatin administration on formalin-induced ulcer forming and healing. (A) Column graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on the incidence of ulcers at day 3 and day 7 after intraplantar formalin injection. (B) Column graphs showing the effect of i.p. ulinastatin (2000, 10,000 and 50,000 U/kg) pre-treatment on ulcers healing at day 10 after intraplantar formalin injection. (C) Column graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on the incidence of ulcers at day 3 and day 7 after intraplantar formalin injection. (D) Column graphs showing the effect of i.p. ulinastatin (50,000 U/kg) post-treatment on ulcers healing at day 10 after intraplantar formalin injection.
Figure 7
Figure 7
Effects of ulinastatin on formalin-induced local and central inflammation. (A) Representative H&E staining of the plantar skin taken near the intraplantar injection site from sham group and formalin group receiving saline or ulinastatin treatment. Scale bars, 50 μm. (B) Quantitative scoring showed ulinastatin treatment dramatically reduced intraplantar formalin injection-induced inflammation infiltration in plantar skin. #P < 0.05 vs Sham, &P < 0.05 vs formalin+saline. (C) Representative immunohistochemistry staining for Iba1 in ipsilateral spinal dorsal horn (L4-L5) from sham group and formalin group receiving saline or ulinastatin treatment. (D) Quantitative analyses showed that ulinastatin treatment significantly reduced the number of activated microglia in formalin group compared to the saline treatment. #P < 0.05 vs Sham, &P < 0.05 vs formalin+saline, one-way ANOVA followed by Tukey’s post hoc test. (E) Representative immunohistochemistry staining for GFAP in ipsilateral spinal dorsal horn (L4-L5) from sham group and formalin group receiving saline or ulinastatin treatment. (F) Quantitative analyses showed that ulinastatin treatment significantly reduced the number of activated astrocyte in formalin group compared to the saline treatment. #P < 0.05 vs Sham, &P < 0.05 vs formalin + saline, one-way ANOVA followed by Tukey’s post hoc test.
Figure 8
Figure 8
Effects of ulinastatin on acetic acid-induced local and central inflammation. (A) Representative H&E staining of the peritoneum tissue taken from sham group and acetic acid group receiving saline or ulinastatin treatment. Scale bars, 50 μm. (B) Quantitative scoring showed ulinastatin treatment dramatically reduced intraperitoneal acetic acid injection-induced inflammation infiltration in peritoneum tissue. #P < 0.05 vs Sham, &P < 0.05 vs acetic acid+saline. (C) Representative immunohistochemistry staining for Iba1 in spinal dorsal horn (T10-L2) from sham group and acetic acid group receiving saline or ulinastatin treatment. (D) Quantitative analyses showed that ulinastatin treatment significantly reduced the number of activated microglia in acetic acid group compared to the saline treatment. #P < 0.05 vs Sham, &P < 0.05 vs acetic acid +saline, one-way ANOVA followed by Tukey’s post hoc test. (E) Representative immunohistochemistry staining for GFAP in spinal dorsal horn (T10-L2) from sham group and acetic acid group receiving saline or ulinastatin treatment. (F) Quantitative analyses showed that ulinastatin treatment significantly reduced the number of activated astrocyte in acetic acid group compared to the saline treatment. #P < 0.05 vs Sham, &P < 0.05 vs acetic acid + saline, one-way ANOVA followed by Tukey’s post hoc test.
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