Effect of shinbuto and ninjinto on prostaglandin E 2 production in lipopolysaccharide-treated human gingival fibroblasts

Previously, we revealed that several kampo medicines which are used for the patients with excess and/or medium patterns [kakkonto (TJ-1), shosaikoto (TJ-9), hangeshashinto (TJ-14), and orento (TJ-120)] decreased prostaglandin (PG)E 2 by LPS-treated human gingival fibroblasts (HGFs). Currently, we examined other kampo medicines which are used for the patients with deficiency pattern [bakumondoto (TJ-29), shinbuto (TJ-30), ninjinto (TJ-32), and hochuekkito (TJ-41)] and the herbs which construct shinbuto and ninjinto using the same experimental model. Shinbuto and ninjinto concentration-dependently decreased LPS-induced PGE 2 production by HGFs, whereas hochuekkito weakly decreased and bakumondoto did not decrease PGE 2 production. Shinbuto and ninjinto did not alter cyclooxygenase (COX) activities and the expressions of molecules involved in arachidonic acid cascade. Next, we examined which herbs constructing shinbuto and ninjinto decrease LPS-induced PGE 2 production. Among these herbs, shokyo ( Zingiberis Rhizoma ) and kankyo ( Zingiberis Processum Rhizoma ) strongly and concentration-dependently decreased LPS-induced PGE 2 production. However, both shokyo and kankyo did not alter the expressions of molecules involved in arachidonic acid cascade. These results suggest that shokyo and kankyo suppress phospholipase (PL)A 2 activity. We demonstrated that kampo medicines for the patients with deficiency pattern may suppress ABSTRACT 11 Previously, we revealed that several kampo medicines which are used for the patients with excess and/or medium patterns [kakkonto (TJ-1), shosaikoto (TJ-9), hangeshashinto (TJ-14), and orento (TJ-120)] decreased prostaglandin (PG)E 2 by LPS-treated human gingival ﬁbroblasts (HGFs). Currently, we examined other kampo medicines which are used for the patients with deﬁciency pattern [bakumondoto (TJ-29), shinbuto (TJ-30), ninjinto (TJ-32), and hochuekkito (TJ-41)] and the herbs which construct shinbuto and ninjinto using the same experimental model. Shinbuto and ninjinto concentration-dependently decreased LPS-induced PGE 2 production by HGFs, whereas hochuekkito weakly decreased and bakumondoto did not decrease PGE 2 production. Shinbuto and ninjinto did not alter cyclooxygenase (COX) activities and the expressions of molecules involved in arachidonic acid cascade. Next, we examined which herbs constructing shinbuto and ninjinto decrease LPS-induced PGE 2 production. Among these herbs, shokyo ( Zingiberis Rhizoma ) and kankyo ( Zingiberis Processum Rhizoma ) strongly and concentration-dependently decreased LPS-induced PGE 2 production. However, both shokyo and kankyo did not alter the expressions of molecules involved in arachidonic acid cascade. These results suggest that shokyo and kankyo suppress phospholipase (PL)A 2 activity. We demonstrated that kampo medicines for the patients with deﬁciency pattern may suppress inﬂammatory responses in addition to those with excess and medium patterns. Moreover, kampo medicines which contain shokyo or kankyo are considered to be effective for the treatment of the inﬂammatory diseases.

Previously, we revealed that several kampo medicines which are used for the patients with excess and/or medium patterns [kakkonto (TJ-1), shosaikoto (TJ-9), hangeshashinto , and orento (TJ-120)] decreased prostaglandin (PG)E 2 by LPS-treated human gingival fibroblasts (HGFs). Currently, we examined other kampo medicines which are used for the patients with deficiency pattern [bakumondoto , shinbuto (TJ-30), ninjinto (TJ-32), and hochuekkito (TJ-41)] and the herbs which construct shinbuto and ninjinto using the same experimental model. Shinbuto and ninjinto concentrationdependently decreased LPS-induced PGE 2 production by HGFs, whereas hochuekkito weakly decreased and bakumondoto did not decrease PGE 2 production. Shinbuto and ninjinto did not alter cyclooxygenase (COX) activities and the expressions of molecules involved in arachidonic acid cascade. Next, we examined which herbs constructing shinbuto and ninjinto decrease LPS-induced PGE 2 production. Among these herbs, shokyo (Zingiberis Rhizoma) and kankyo (Zingiberis Processum Rhizoma) strongly and concentration-dependently decreased LPS-induced PGE 2 production. However, both shokyo and kankyo did not alter the expressions of molecules involved in arachidonic acid cascade. These results suggest that shokyo and kankyo suppress phospholipase (PL)A 2 activity. We demonstrated that kampo medicines for the patients with deficiency pattern may suppress inflammatory responses in addition to those with excess and medium patterns. Moreover, kampo medicines which contain shokyo or kankyo are considered to be effective for the treatment of the inflammatory diseases.

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Periodontal disease is an inflammation disease of the gingiva and destroy periodontal 33 tissues. In severe case, alveolar bone is absorbed. In inflammatory responses and tissue 34 degradation, prostaglandin E 2 (PGE 2 ), interleukin (IL)-6, and IL-8 play important roles. 35 Because PGE 2 has several functions in vasodilation, the enhancement of vascular per-36 meability and pain, and osteoclastogenesis induction, PGE 2 participate in inflammatory 37 responses and alveolar bone resorption in periodontal disease (Noguchi and Ishikawa, 2007). 39 Previously, we reported that several kampo medicines, shosaikoto (TJ-9) (Ara et al., induced PGE 2 production by human gingival fibroblasts (HGFs). Moreover, we showed 43 shokyo, kanzo, and keihi, which are herbs contained in kakkonto, decrease PGE 2 44 production (Ara and Sogawa, 2016). These results suggested that these kampo medicines 45 and herbs have anti-inflammatory effects in periodontal disease.

Effects of kampo medicines on HGFs viability
147 First, we examined the effect of four kampo medicines (bakumondoto, shinbuto, ninjinto, 148 and hochuekkito) on HGFs viability. Bakumondoto did not affect the viability up to 10 149 mg/ml at 24 h treatment ( Fig. 1). In contrast, Shinbuto, ninjinto, and hochuekkito did 150 not affect the viability up to 2 mg/ml but decreased at 5 mg/ml and 10 mg/ml (Fig. 1).

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Therefore, up to 1 mg/ml of kampo medicines was used in further experiments because

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Effects of shinbuto and ninjinto on arachidonic acid cascade 169 To clarify the mechanism that shinbuto and ninjinto decreased LPS-induced PGE 2 170 production more directly, we examined the effects of these two kampo medicines on 171 arachidonic acid cascade. First, we examined the effect of shinbuto and ninjinto on 172 COX activity. In order to bypass PLA 2 , we added exogenous arachidonic acid to HGFs 173 treated with LPS alone or LPS plus kampo medicine (shinbuto or ninjinto). Then, we 174 measured PGE 2 level produced by COX. However, shinbuto and ninjinto did not affect

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In the recent study, we showed that shokyo suppressed LPS-induced PGE 2 produc-239 tion by HGFs and that shokyo may suppress PLA 2 activity (Ara and Sogawa, 2016). In 240 the present study, we examined the effect of kankyo in comparison with shokyo. We 241 showed that shokyo and kankyo only slightly increase cPLA 2 expression but did not 242 alter annexin 1 expression (Fig. 7). Moreover, we showed that shinbuto and ninjinto, 243 which contain shokyo and kankyo respectively, did not altered PGE 2 production when 244 arachidonic acid was added to bypass their upstream pathway (Fig. 3). These data 245 suggest that shokyo and kankyo did not affect the downstream pathway of arachidonic 246 acid, which include COX-2 and PGE synthase. In addition, shinbuto and ninjinto did 247 not affect ERK phosphorylation (Fig. 5). From our findings described above could 248 not explain the mechanism to decrease PGE 2 production. Because gingerols in ginger 249 6/12 are reported to inhibit both calcium-independent PLA 2 (iPLA 2 ) and cPLA 2 activities 250 (Nievergelt et al., 2011), shokyo and kankyo are suggested to inhibit PLA 2 as discussed 251 in previous study (Ara and Sogawa, 2016). Previously, we showed that cPLA 2 is the 252 main isoform in HGFs (Ara and Sogawa, 2016) among the subtypes such as cPLA 2 , 253 iPLA 2 , and secretory PLA 2 (sPLA 2 ) (Burke and Dennis, 2009). Therefore, shokyo and 254 kankyo may mainly inhibit cPLA 2 activity in HGFs. Previously, we reported that orento 255 decreases LPS-induced PGE 2 production via the suppression of ERK phosphorylation 256 (Ara et al., 2010). However, orento also may decrease LPS-induced PGE 2 production 257 by inhibition of cPLA 2 activity because orento contains kankyo. 258 We demonstrated that shokyo and kankyo concentration-dependently decreased shogaols may be different among the kinds of cells.

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We demonstrated that shinbuto and ninjinto decreased LPS-induced PGE 2 production 298 by HGFs. Also shokyo and kankyo, which are included in these kampo medicines 299 respectively, concentration-dependently decreased LPS-induced PGE 2 production. How-300 ever, shokyo and kankyo did not the expression of the molecules in arachidonic acid 301 cascade, suggesting that shokyo and kankyo inhibit cPLA 2 activity. Therefore, the 302 kampo medicines that contain shokyo or kankyo may have the ability to decrease PGE 2 303 production. We demonstrate that the kampo medicines used for the patients with defi-304 ciency pattern have anti-inflammatory effects as well as those with excess pattern and 305 medium pattern. We expect that kampo medicines are used for the improvement of 306 inflammatory diseases such as periodontal disease and stomatitis in patients with any 307 pattern.

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This study was approved by the Ethical Committee of Matsumoto Dental University 310 (No. 0063).

Figure 7
462 Effects of shokyo and kankyo on cPLA 2 , annexin 1, and COX-2 expressions. HGFs 463 were treated with a combination of LPS (0 or 10 ng/ml) and herb (1 mg/ml) for 8 h, and 464 protein levels were examined by Western blotting. s, shokyo; k, kankyo.  The ingredient of shinbuto formula  The ingredient of ninjinto formula

Figure 1(on next page)
Effects of kampo medicines on the cytotoxicity.