JP3823030B2 - Preventive and / or therapeutic agent for lupus erythematosus - Google Patents

Description

【００７３】
以上の結果から、冬虫夏草より単離された純粋な天然物Ｈ１−Ａは、インビトロではＰＨＡに活性化された末梢血単核球細胞よりもＩＬ−２の産生を抑制することができた。MRL lpr/lpr動物モデルでは、リンパ節の増殖を阻止し、抗ds-DNA自己抗体の産生を抑制することもできた。またMRL lpr/lprマウスの生存期間を延長することもできた。同時に、腎糸球体のメサンギウム増殖を抑制し、蛋白尿の進行を遅らせることができたが、驚くことに、本研究においては、免疫複合体の沈着に有意な変化が見られなかった。本研究の結果は、この化学構造が明確な単離された天然物が、MRL lpr/lprマウスの自己免疫病を治療する潜在力を持っていることを明らかにした。その他、血清中の抗ds-DNA自己抗体濃度と、腎臓組織病理変化と腎臓免疫複合体沈着との３者間は関連していないが、将来的に、全身性エリテマトーデス性腎炎の原因に対し、より多い検討空間を提供することが可能となる。
【００７４】
本発明のインビトロ試験結果は、Ｈ１−Ａが、ＰＨＡによって活性化された末梢血単球細胞によるＩＬ−２の産生を有効に抑制することが可能であることを示した。この薬物の効果は、周知の免疫抑制剤であるシクロスポリンＡと似ている。前述したように、冬虫夏草の原始複合物であるＨ１−Ａは、細胞免疫に対し、免疫抑制の作用を持っている。この結果は、シクロスポリンＡの皮膚移植における結果と類似している。換言すれば、Ｈ１−Ａは免疫調節剤と呼ばれたほうがよりふさわしい。シクロスポリンＡは、ヒトの全身性エリテマトーデスの治療に有効であると言われている。シクロスポリンＡは、Ｔリンパ球に対し、選択的に使用されている。したがって、Ｔ細胞の反応を主とする病変への治療に用いられる。シクロスポリンＡは、いくつかの抗体と関係している反応にも抑制作用があるが、その主な作用は、Ｔヘルパー細胞より生じた反応に対するものである。比較すると、シクロスポリンＡは、抗原が直接Ｂ細胞を刺激して生じた抗体反応に対する作用が少ない。Ｈ１−Ａは、抗ds-DNA抗体に対する抑制作用が顕著に見られるため、シクロスポリンＡと類似の作用メカニズムを持っているかもしれない。それは、抗ds-DNAの産生が、抗原の起動によるＴ細胞調節の過程であるためである。Ｈ１−Ａは、Ｔ細胞増殖を主な症状とするリンパ節増殖にも抑制作用を有する。全身性エリテマトーデスのメカニズムに関する新観点は、ＣＤ４^＋Ｔ細胞が、MRL lpr/lprマウス自己免疫疾患発生の主因であることである。したがって、現在、このような病気になる原因は、Ｔ細胞の機能のバランスが崩れたか、またはＴ細胞およびＢ細胞間の協力に欠如があるためである。抗ＣＤ３、抗ＣＤ４、および抗ＣＤ８モノクローナル抗体は、マウスのエリテマトーデスの治療で有効であると報告されている。したがって、Ｈ１−Ａ治療効果は、Ｔ細胞の産生を抑制すること、またはＴｈ１／Ｔｈ２の産生を抑制することによって発揮すると推測されている。
【００７５】
Ｔｈ１およびＴｈ２への抑制作用により、マウス体内での抗ds-DNA抗体の産生が顕著に下がった。腎臓局部Ｔｈ１の産生が抑制されたことによるものかもしれない。腎糸球体内のメサンギウム細胞の増殖はこれによって停止した。これらの仮説を、将来の研究でさらに証明することが必要である。本発明は、腎臓切片に免疫複合体沈着の変化が見られないことについて、３つの可能な解釈を提供した。
【００７６】
第１に、腎臓組織病理変化と免疫複合体沈着との間の不関連性は、臨床上で良く見られる。したがって、免疫複合体で全身性エリテマトーデス性腎炎の全ての現象を解釈するのが不可能である。その他の介入可能な因子は、細胞因子または化学因子を含んでいる。したがって、Ｈ１−Ａのメサンギウム増殖への抑制作用は、免疫複合体沈着を下げる以外のメカニズムを通じて発揮しているかもしれない。
【００７７】
第２に、既に沈着した免疫複合体の除去は、多くの時間を要する。ＩＶ類全身性エリテマトーデス性腎炎の臨床経験によると、患者がシクロホスファミドの治療を受けた後、疾病の症状の軽減に伴い、血液中の抗ds-DNA抗体値が先に低下する。腎臓組織中の免疫複合体の排除は、臨床の症状改善よりも遅い。通常、最低６ヶ月の治療を経過した後、腎臓組織に顕著な変化が初めて見られる。
【００７８】
第３に、Ｈ１−Ａは、マクロファージの、免疫複合体を排除する能力を改善しない。以上、Ｈ１−Ａは、ヒト全身性エリテマトーデスを治療するための潜在的な単離された天然物である。Ｈ１−Ａは、シクロスポリンＡよりも安全性が高い。冬虫夏草が中国伝統的な治療に用いられるのは、千年以上の歴史を持っているにもかかわらず、今までに、腎臓毒性に関する報告が全くなかったからである。反対に、冬虫夏草は、シクロスポリンＡおよびアミノグリコシドによってもたらされた腎臓毒性を有効に避けることができると報告されている。本研究も、このような観点を証明した。腎臓切片には、いかなる腎臓毒性を全く見ることがなく、逆にＨ１−ＡがMRL lpr/lprマウスの腎臓機能に対し、保護作用さえ有している。
【００７９】
免疫のメカニズムから見ると、最近、全身性エリテマトーデス患者のＴ細胞機能に欠陥があると多くの文献に述べられている。その中に、ＩＬ−２の産生が減少し、かつＩＬ−２によって誘発されたＴ細胞増殖の反応も顕著に下がることが含まれている。Gutierrez-Ramosらは、ＩＬ−２／ワクシニアの組換えワクチンで、MRL lpr/lprマウスの治療に成功した。いくつかのいわゆる免疫調節剤は、ＩＬ−２による欠陥を修正し、胸腺内の細胞分裂および末梢Ｔ細胞の活性を促進するといわれてきた。しかし、ある人は、完全に反対な意見を提出した。Cuadradoらは、全身性エリテマトーデス患者の血液中にＩＬ−４、ＩＬ−２、およびｓＩＬ−２Ｒの比較的正常かつ顕著な上昇が見られると報告した。これに対し、Razらは、ＩＬ−２ ｃＤＮＡをMRL lpr/lprマウスの体内に導入することで、その病状を悪化させ、さらに、自己抗体の産生も促進すると報告した。発明者らの結果は、後者の意見とほぼ一致している。インビトロで、Ｈ１−Ａは、末梢単核球細胞でのＩＬ−２の産生を抑制する；MRL lpr/lprマウス動物モデルでは、Ｈ１−Ａは、臨床の症状および腎臓の組織病変を改善することができる。
【００８０】
以上のように、本発明は、中国の伝統的な漢方薬である冬虫夏草から分離された、明確な構造を有する単離された天然物Ｈ１−Ａを用いて、自己免疫疾患モデルのMRL lpr/lpr体内の治療効果を上げることに成功した。
【００８１】
実施例３：誘発ＩｇＡ腎炎を持つマウスに対する冬虫夏草抽出物（１％および0.5％のＦ２）の効果
【００８２】
１．ＩｇＡ腎炎を誘発する方法：使用される抗原はＲ３６Ａであり、抗体はａ−Ｒ３６Ａ−ＩｇＡ−ｍＡｂである。まず、Balb/cマウスの腹部に２mgの抗体を注射し、２時間後、尾静脈に100μgの抗原を注射した。次いで、24時間ごとに100μgの抗原を注射し（１週間連続）、これにより、免疫複合体が腎間質に沈着し、最初の注射後約５日間、血尿および蛋白尿が現れた。
【００８３】
２．ＩｇＡ腎炎を誘発するマウスを、投薬群（ｎ＝６）および対照群（ｎ＝６）に分けた。投薬群は、１％および0.5％のＦ２を摂取させた。毎日、各マウスに約3ｇの正常飼料を摂取させた。試験群と対照群とのマウスを、毎日、血尿および蛋白尿を測定し、７日後、マウスを屠殺した。その腎臓を取り、組織病理学検査および免疫蛍光検査を行い、そして血清中のＡＬＴ、ＡＳＴ、および肝臓中のコレステロール、チトクロムＰ４５０の変化を観察した。
【００８４】
Ｉ．血尿および蛋白尿の測定：
尿液試験紙（Urine Strip）で検査した。
【００８５】
ＩＩ．腎臓組織病理学検査：
腎臓組織を10％中性ホルマリンで固定し、漸増濃度のアルコールで脱水した後、パラフィン包埋し、厚さ４μmの切片を切り、ヘマトキシリン-エオシンで染色し、光学顕微鏡にて観察した。各マウスについて50個の糸球体を観察し、糸球体の破壊程度を５等級に分け、点数が高ければ、糸球体の破壊程度が高くなる。最後に点数を累計し、総数（Total）で表示する。
【００８６】
ＩＩＩ．免疫蛍光検査：
腎臓組織をO.C.T.compoundで包埋し、−70℃の冷凍庫に保存した。免疫染色を行う際に、冷凍切片機で４μmの組織切片を切り出し、そしてアセトンで固定し、次いで、蛍光標識されたヤギ抗マウスＩｇＡ、ヤギ抗マウスＣ３、およびヤギ抗フィブリノーゲンで直接免疫蛍光の染色を実施し、蛍光顕微鏡で観察した。蛍光染色強度を４等級に分け、点数が高ければ、蛍光の強度が強くなり、ＩｇＡ、ＩＣ沈着およびＣ３補体の活性化が高くなることを表す。
【００８７】
３．統計方法：
血尿、蛋白尿、組織病理学検査および免疫蛍光検査は、χ２乗検定で分析を行った。血清中のＡＬＴ、ＡＳＴおよび肝臓中のコレステロール、チトクロムＰ４５０の統計は、Studentのｔ検定で分析を行った。P＜0.05は、統計学的に有意であるとみなす。
【００８８】
結果：
１．血尿および蛋白尿を表２に示す。
【００８９】
【表２】

【００９０】
抗原を注射した５日後、血尿および蛋白尿が現れた。７日目に、１％Ｆ２群および0.5％Ｆ２群では、血尿および蛋白尿の顕著な軽減が見られた（P＜0.05）。
【００９１】
２．腎臓組織病理学検査の結果を表３に示す。
【００９２】
【表３】

【００９３】
総数（Total）から見ると、１％Ｆ２群は、腎糸球体の破壊程度が有意に軽減したが（P＜0.05）、0.5％Ｆ２群では、統計学的有意差が認められなかった。
【００９４】
３．免疫蛍光検査の結果を表４および５に示す。
【００９５】
【表４】

【００９６】
【表５】

【００９７】
総数（Total）評価より、投薬の１％Ｆ２群および0.5％Ｆ２群は、対照群と比較すると、ＩｇＡ ＩＣ沈着またはＣ３補体の活性化の両方とも、薬剤量に比例して軽減されていた。１%Ｆ２群は、対照群と比較すると、統計学的有意差が認められた（P＜0.05）。
【００９８】
４．肝臓機能検査の結果を表６に示す。
【００９９】
【表６】

【０１００】
肝重量および体重について、投薬群と対照群とは、すべて差がなかった：100倍の肝重量対体重の比率から見ると、１％Ｆ２群は6.75±0.09であり、対照群は7.12±0.12であった。１％Ｆ２は肝重量は明らかに減少した（P＜0.05）。血清中のＡＬＴおよびＡＳＴは、投薬群と対照群との間に、顕著な変化がなかった。肝臓中のコレステロールの含有量は、投薬群と対照群との間に、統計学的有意差がなかった。肝臓中のチトクロムＰ４５０の含有量は、１％Ｆ２群が0.31±0.03nmol/mgタンパク質であり、対照群は0.23±0.01nmol/mgタンパク質であった。１％Ｆ２は、チトクロムＰ４５０の含有量を顕著に増加させた（P＜0.05）。肝重量対体重の比率から見ると、急性毒性試験では、２％Ｆ２を投与すると、肝臓腫脹が見られた。ＩｇＡ腎炎が誘発された動物モデルでは、１％Ｆ２を投与すると、肝臓の萎縮が見られた。これらは反対の結果を示している。急性毒性試験は、正常ICRマウスを対照群とし、その100倍肝重量対体重の比率は5.44±0.17であった。ＩｇＡ腎炎が誘発されたBalb/cマウス試験では、正常の飼料の飼育を対照群とした。予めＩｇＡ腎炎が誘発された動物自体は、その100倍肝重量対体重の比率が7.12±0.12であり、ＩｇＡ腎炎のマウスには、肝臓腫脹の現象が見られる。２つの試験に使用された動物は異なる品種であった。さらに、正常のBalb/cマウスに、「冬虫夏草抽出物で飼育する」方式で、正常飼料を飼育すると、最後には、100倍肝重量対体重の比率は5.50±0.24となり（図７を参照のこと）、これは、ICRマウスの正常な100倍肝重量対体重の比率（5.44±0.17）と一致していた。これは、ＩｇＡ ＩＣ自体が、顕著な肝臓腫脹現象を引き起こすことを示した（P＜0.05）。そのメカニズムは不明である。ただし、１％Ｆ２を投与すると、ＩｇＡ ＩＣ誘発の肝臓腫脹が有意に軽減されたが（P＜0.05）、正常値までに回復することができなかった。
【０１０１】
実施例４：体外でＨ１−Ａの細胞に対する安全性
Ｈ１−Ａの細胞に対する活性および毒性について検討し、その結果を図８に示した。最上方の図８（ａ）は、トリパンブルー染色で細胞の活性を測定した結果であり、72時間培養した３群（ヒトメサンギウム細胞のみ、ヒトメサンギウム細胞＋ＩＬ−１とＩＬ−６、およびヒトメサンギウム細胞＋ＩＬ−１とＩＬ−６および40μM/mlのＨ１−Ａ）の細胞活性に差異がないことを示した。死滅した細胞がLDHを放出するため、同じ(ａ)試験で、72時間後、培養液中のLDHの濃度を測定し、その結果を図８（ｂ）に示した。３群は、共に統計上の差異がなかった。細胞が生存すれば、メサンギウム細胞も増殖する。そこで、ＭＴＴ方法で、細胞増殖の状況を分析した。その結果を図８（ｃ）に示した。３群は共に統計上の差がなかったことが分かる。
【０１０２】
実施例５：ＩｇＡ腎炎が誘発されたマウスに対するＨ１−Ａの影響
実施例３で誘発したＩｇＡ腎炎マウスに、10mg/mlのＨ１−Ａを、腹部に１回注射し、７日間連続して注射した。
【０１０３】
治療群のＩｇＡ腎炎マウスとコントロール群の血尿／蛋白尿に対する影響を比較した。表７に示した結果から、治療群の血尿／蛋白尿が顕著に改善され、かつ統計学的に有意であると分かる。
【０１０４】
腎臓組織の病理変化について、コントロール群では、重篤なメサンギウム増生およびメサンギウム細胞増殖が見られたが、治療群はＨ１−Ａの治療を受けた後、組織病理変化の顕著な改善が見られた。治療群の腎臓切片は、ヘマトキシリン-エオシン染色で、僅かなメサンギウム増殖があっただけであり、蛍光染色の改善は表７に示されている。治療群には顕著な改善があり、かつ統計学的に有意である。
【０１０５】
【表７】

【０１０６】
【発明の効果】
冬虫夏草より分離されたＦ２は、体外において、腎間質細胞の活性化を抑制することが可能で、体内において、ＩｇＡ腎炎の発生または悪化を阻止することができる。Ｆ２は急性毒性がほとんどなく、かつＩｇＡ ＩＣより誘発された肝臓腫脹を軽減することもできる。Ｆ２より単離された活性天然物（Ｈ１−Ａ）は、「体内ＩｇＡ腎炎の発生」を阻止する動物モデルで、良好な治療効果を持っている。
【０１０７】
本発明は、好ましい実施例について説明しているものの、それらの種々の変更は、この明細書を読めば、当業者に明かなことが理解されるべきである。従って、ここで開示の発明は、添付の請求の範囲に入るようなこれらの変更を含むべく意図されていることが理解されるべきである。
【図面の簡単な説明】
【図１】 冬虫夏草から活性画分と活性化合物とを分離する工程を示すフローチャートである。
【図２】 本発明のＨ１−Ａが、末梢血単核球細胞によるＩＬ−２の産生に対する影響を示すグラフである。●：Ｈ１−Ａのみ ；▽：単核球細胞をＰＨＡ（10μg/ml）＋Ｈ１−Ａで刺激。
【図３】 Ｈ１−ＡのMRL lpr/lprマウスの生存期間に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図４】 本発明のＨ１−ＡのMRL lpr/lprマウスのリンパ病変に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図５】 本発明のＨ１−ＡのMRL lpr/lprマウスの蛋白尿に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図６】 本発明のＨ１−ＡのMRL lpr/lprマウスの腎臓機能に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図７】 本発明のＨ１−Ａの、MRL lpr/lprマウスにおける抗ds-DNA自己抗体の産生に対する影響を示すグラフである。■：Ｈ１−Ａを投与した治療群；○：未治療の対照群。
【図８】 本発明のＨ１−Ａの細胞活性に対する影響を示すグラフである。■：対照群で、ヒトのメサンギウム細胞のみ；□：ヒトのメサンギウム細胞＋ＩＬ−１、ＩＬ−６；□：ヒトのメサンギウム細胞＋ＩＬ−１、ＩＬ−６およびＨ１−Ａ 40μM/mlの72時間の培養。（ａ）トリパンブルー染色による細胞の活性の観察；（ｂ）放出されたLDHの測定による細胞の死亡の観察；（ｃ）MTT試験による細胞の増殖の観察。[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to a pharmaceutical composition comprising an active fraction of Cordyceps sinensis (Berk) Sacc and an active compound and a method for improving renal function using the same. The present invention also relates to methods for preventing and / or treating lupus erythematosus and IgA nephritis.
[0002]
[Prior art]
  Lupus erythematosus nephritis is a multi-organ erosive and systemic inflammatory chronic disease. The disease is associated with immune system disorders and a wide distribution of immune complexes. Autoantibodies against many autoantigens are found in the body of patients with systemic lupus erythematosus. These autoantigens are cell membranes, cell nuclei, or cytoplasmic tissue components, and the autoantibodies produced include antinuclear antibodies, anti-ds-DNA antibodies, anticardiolipin antibodies, anti-Ro antibodies, anti-La antibodies, etc. . A clinical feature of systemic lupus erythematosus is that the patient repeats the process of onset and alleviation. However, its etiology has not yet been found. Therefore, clinical diagnosis of systemic lupus erythematosus is currently made based on the 1982 “Revised Rheumatism Association Criteria for SLE” (Tables I-I). The pathogenesis of systemic lupus erythematosus is currently presumed to be related to genetic inheritance, environmental factors, foreign antigens (eg, viruses), hormones and stress. The symptoms are divided into systemic lesions or local lesions. Systemic lesions refer to the following conditions: That is, immune regulation is unbalanced, and B cells are automatically proliferated and activated by polyclonal cells without being regulated by T cells, resulting in a large amount of autoantibodies. Alternatively, there is an imbalance between both Th1 / Th2 T cells and a defect in T cell resistance, which may lead to B-cell expansion, somatic mutation, and transformation by Ag-driven T cell induction. Brought about. In both cases, many autoantibodies and immune complexes are produced. The deposition of these immune complexes induces local lesions. Therefore, in the body of patients with systemic lupus erythematosus, immune complex deposition is observed in each organ, and at the same time, an inflammatory reaction jointly induced by cytoplasm, adhesion molecules, and inflammatory cells is observed.
[0003]
  The difference in systemic lupus erythematosus between children and adults is 1:10 for children compared to 1:10 for adults. Other than that, pre-pubertal children rarely have typical skin symptoms such as erythema and photosensitization, but blood (eg hemolytic anemia, thrombocytopenia) or kidney (proteinuria, hematuria) , Nephritis syndrome) is relatively common. When nephritis is severe, especially when severe lupus erythematosus nephritis is associated, the prognosis is poor, resulting in relatively high morbidity and mortality.
[0004]
  In the classification of lupus erythematosus by the World Health Organization (WHO), lupus erythematosus is classified into 5 categories. Groups I through IV are classified based on the severity of mesangial cell proliferation and the degree of glomerulosclerosis. Class V, unlike other classes in pathological changes and clinical symptoms, can be a simple membranous glomerular change, but can also be a pathological change in which symptoms of Group II, III and IV coexist. Of these five types of nephritis, class IV is so-called membranoproliferative glomerulonephritis (MPGN), which is nephritis with the poorest prognosis. MPGN pathological sections show prominent features such as endothelial cell proliferation, mesangial cell proliferation, capillary wall thickening, vascular embolism, diffuse cell necrosis, inflammatory cell infiltration, crescent-forming glomerulosclerosis, atrophy and fibrosis Symptoms are seen. Among these lesions, for example, mesangial cell proliferation, leukocyte infiltration, fibrous necrosis, vitreous thrombus and interstitial inflammation are related to the activity of the disease and are called activity indexes Yes. In addition, glomerulosclerosis, fibrotic meniscus, renal tubule atrophy and interstitial fibrosis are chronic and unrecoverable lesions and are called chronicity indexes. The activity of the kidney section and the high chronic index indicate the extent of nephritis and predict the likelihood that the patient's symptoms will progress to end-stage nephritis.
[0005]
  In the pathogenesis of systemic lupus erythematosus nephritis, glomerular mesangial cells, glomerular epithelial cells, and renal matrix all play an important role in acute glomerular lesions and glomerulosclerosis. Among them, the action of mesangial cells is leading. Mesangial cells are very interesting cells, 95% of which are derived from endogenous kidney cells. Its function is similar to that of vascular wall muscle cells and macrophages. The other 5% originates from bone marrow cells and moves into the glomerulus during embryo development. The activation of mesangial cells is highly related to the onset mechanism of membranoproliferative glomerulonephritis and glomerulosclerosis. According to the literature, mesangial cells are activated by effects such as infection, stimulation of immune complexes, changes in hemodynamics, metabolite and fat accumulation, or proliferation of residual kidney tissue due to loss of kidney tissue. In the pathogenesis of systemic lupus erythematosus nephritis, these external stimuli produce immune complex deposits formed by antigens and autoantibodies. Mesangial cells release cytokines, growth factors, and adhesion molecules after being activated by immune complexes. These substances have autocrine, paracrine, and endocrine functions that not only lead to regrowth and reactivation of mesangial cells in the glomerulus, but also to other cells in the kidney (eg, renal epithelial cells) Reactivate tubule cells, vascular endothelial cells). Due to the activation of mesangial cells, renal epithelial cells, and tubular cells, the kidney secretes more and more cytokines, growth factors, adhesion molecules, and more fibrocytes and inflammatory cells (eg, macrophages, B cells, T cells) and activated complement. These endogenous kidney cells, exogenous inflammatory cells, fibrocytes and vascular endothelial cells interact to gradually accumulate mesangial matrix, and finally to glomerulosclerosis, tubular atrophy, and fibrosis of the renal interstitial tissue To develop.
[0006]
  In fact, systemic lupus erythematosus and glomerular sclerosis are fairly complex processes, but mesangial cells and renal epithelial cells also play a significant role. The whole process is divided into three stages according to cause, time and order. The first step is the deposition of immune complexes. The second stage is the activation of mesangial cells, renal epithelial cells and a series of cytokine network changes induced thereby. At this stage, mesangial cells have the following changes: 1. Accelerate cell turnover; 2. changes in cell appearance; 3. changes in the secretory form of the cells; 4. release some inflammation-causing medium; and Accelerates the synthesis of the renal matrix. The third stage is that mesangial cells provide fibrocytes from secretory cell factors and adhesion molecules, and at the same time stimulate angiogenesis. Addition of mesangial cell proliferation, renal matrix synthesis, tissue fibrosis, and angiogenesis leads to glomerulosclerosis.
[0007]
   According to clinical experience and the MRL lpr / lpr systemic lupus erythematosus animal model, it is difficult to prevent or eliminate immune accumulation of systemic lupus erythematosus nephritis (ie, a first stage renal lesion) within a short period of time. Therefore, a relatively feasible treatment strategy would prevent the second stage mesangial cell proliferation, activation, secretion and renal matrix synthesis and avoid entering the kidney into the third stage glomerulosclerosis That is.
[0008]
  To date, the treatment of systemic lupus erythematosus nephritis has been immunosuppressive or immunomodulatory treatment with drugs for the whole body, including corticosteroids (corticosteroids), high dose methylprednisolone, cyclodextrin. Includes hormonal therapies such as phosphamide, azathioprine, MTX, the antimalarial drug prenyl, non-steroidal anti-inflammatory drugs, high doses of immunoglobulin or dehydroepiandrosterone (DHEA), tamoxifen. In recent years, cyclosporine, FK506 has been increasingly used for the treatment of systemic lupus erythematosus nephritis.
[0009]
  However, the above treatment methods cannot completely and effectively suppress the progression of systemic lupus erythematosus nephritis, and non-selective immunosuppression can always cause clinically fatal complications. Many literatures have proposed different treatment plans. One such treatment model is to convert systemic immunotherapy for systemic lupus erythematosus nephritis to local treatment. There, heparin treatment improves renal local blood flow; the ACE inhibitor captopril suppresses angiotensin II production; an ornithine carboxylase inhibitor suppresses renal nitric oxide production. These therapeutic effects have been demonstrated in animal models of mice.
[0010]
  Another therapeutic model is to selectively perform immunotherapy due to the pathogenesis of systemic lupus erythematosus nephritis. As described above, systemic lupus erythematosus has been disrupted in the T cell mechanism and can cause a lack of cooperation between T cells and B cells or an imbalance between Th1 / Th2. The literature shows the effects of neonatal thymectomy on animal models of systemic lupus erythematosus mice against homing receptor monoclonal antibodies, IL-2 / vaccinia recombinant vaccines, transgenic T cell gene transfer, etc. It has been reported that it can. However, the use of these treatments in humans must be carefully considered in terms of technology and complications. Therefore, the future treatment method for systemic lupus erythematosus nephritis is to clearly understand the cause of its progressive deterioration, thereby enabling appropriate treatment at the appropriate time and avoiding unnecessary systemic side effects. .
[0011]
  On the other hand, in clinical practice of IgA nephritis, paroxysmal hematuria and / or proteinuria always appeared in a chronic disease manner. The cause of IgA nephritis is that IgA immune complexes are deposited on the glomerular mesangial and activate mesangial cells. The activated mesangial cells release interleukin 1 (IL-1), interleukin 6 (IL-6), tumor necrosis factor α (TNFα), and proliferate mesangial cells. Proliferated mesangial cells release IL-1, IL-6, platelet derived growth factor (PDGF), transforming growth factor β (TGF-β) and the like. The role of these cellular hormones and growth factors is the same as autocrine and is a vicious cycle of mesangial growth,₂ ⁻, H₂O₂, Platelet activating factor (PAF), prostaglandin E₂(PGE₂), Thromboxane B₂(TXB₂), And neutral proteases can be released. Thus, mesangial growth hardens the glomeruli and damages the renal basement membrane. In humans, after getting sick, about 20% gradually progress to uremia. To date, the disease has not been prevented with appropriate therapeutic agents. Therefore, researching therapeutic drugs is the most important issue.
[0012]
  An immunomodulator is a therapeutic agent with potential, and can suppress immune enhancement and simultaneously treat immunodeficiency. There are many Chinese medicines with immunomodulatory functions in China. One of them is the famous Cordyceps.
[0013]
  Cordyceps Sinensis (Berk) Sacc is a complex of centripetal and larval carcasses in which ergot fungi belonging to the genus Cordycepsae are parasitic on larvae of bat insects (Hepialus armoricanus Oberthur) as categorized as follows: is there. Since the host is a larva of insects such as Lepidoptera and Coleoptera, the hyphae invade the larvae in the soil in the winter, fill them with mycelia, and die; Come out. Therefore, it is said to be “Food Cordyceps”. Its production areas are Sichuan, Yunnan, Qinghai, Tibet, etc., but the price is very high because it is difficult to acquire.
[0014]
Taxonomy
  Fungi
    Ascomycotina
      Pyrenomycetes
        Sphaeriales
          Clavicipitaceae
            Cordyceps
[0015]
  Reports on the therapeutic effects of Cordyceps are as follows.
[0016]
(1) Effects on the immune system
  Cordyceps is said to have a bidirectional regulatory function for cell immunity. In 1990, Zhang et al. Reported that water extract of Cordyceps can promote thymocyte mitosis and further T lymphocyte proliferation in terms of promoting cellular immunity. In 1985, Zhang et al. Also reported that the extract could activate mouse peritoneal macrophages. The combination of Cordyceps sinensis and peach nuts can cure hepatitis and cirrhosis, and can further increase T lymphocyte conversion rate, natural killer cell function, and CD4 / CD8 cell ratio (Zhu 1992). Cordyceps has a role of stimulating division in splenic lymphocytes, stimulates spleen cells of chronic renal failure rats, increases IL-2, further lowers blood urea nitrogen and creatinine levels, Can be raised. The results indicate that Cordyceps has a regulatory function on cellular immunity in rats with chronic renal failure (Cheng Q, 1992). In addition, Cordyceps can protect T helper cells, increase the value of Th / Ts, and counteract the immunosuppressive action of steroids and cyclophosphamide. Therefore, Cordyceps is presumed to be a kind of immunomodulator or biological response modifier for cellular immunity. Thus, Chen et al. Reported in 1991 that Cordyceps can treat patients who have been immunocompromised or acquired immunosuppressed.
[0017]
  On the other hand, Cordyceps has the effect of suppressing cellular immunity. Zhang et al. Reported in 1990 that Cordyceps mycelium could inhibit mouse peripheral leukocyte uptake and spleen lymphocyte proliferation. The extract inhibits the excretion of the xenograft heart or prolongs organ survival.
[0018]
  Cordyceps has the same bidirectional regulatory effect on humoral immunity, can promote humoral immunity, directly induce the proliferation of Thy-1 cells, or increase the antibody content of IgG and IgM. . However, Zhu et al. Pointed out in 1992 that Cordyceps sinensis also has an inhibitory effect on humoral immunity. Zhu et al. Found that Cordyceps sinensis had an immune effect similar to cyclosporin A (5 mg / kg) in 1990, although it was found to reduce IgG and IgA during observation of treatment of liver sclerosis after hepatitis. I also pointed out.
[0019]
(2) Effects on kidney function
  Cordyceps can avoid damage to the proximal tubules of gentamicin. One of its actions is to promote cell repair. In addition, cordyceps significantly reduces the degree of acute tubular injury in rats or restores early damage to kidney function in rats with renal failure in rats with acute renal failure model induced by kanamycin I discovered that. The cause is to reduce acute damage to the solvate of tubule cells and cell membrane Na⁺, K⁺-May be related to ATPase. This effect is related to reducing cellular lipid peroxidation (Zhen, 1992). Cordyceps can reduce mortality and lower blood urea nitrogen and serum creatinine levels in rats with chronic renal dysfunction; its clinical application is clearly seen to improve the renal status of renal failure. In addition, Cordyceps can improve nephrotoxicity caused by cyclosporine and improve kidney function.
[0020]
(3) Antitumor activity of Cordyceps
  Cordyceps medicinal solution and artificially cultivated cordyceps mycelium solution have an inhibitory effect on mouse sarcoma 180 and can also enhance the anticancer effect of cyclophosphamide. Cordyceps and artificially-grown cordyceps mycelium have all significant inhibitory effects on the primary growth and spontaneous lung metastasis of Lewis lung cancer transplanted subcutaneously in mice.
[0021]
  The possible mechanism is that cordycepin contained therein suppresses DNA and RNA synthesis, suppresses nucleic acid methylation, suppresses protein kinase activity, and promotes cell differentiation. Has anticancer activity against cancer, colon cancer, and lung cancer.
[0022]
(4) Action on cardiovascular system
  Wild Cordyceps is effective against arrhythmias in experimental animal models. Artificial cordyceps mycelium has the role of significantly increasing coronary blood flow in anesthetized dogs, increasing the resistance of coronary arteries, brain and peripheral blood vessels, and lowering blood pressure. be excited. Its active ingredient is adenosine, which has a direct relaxing action on vascular smooth muscle, which is the main mechanism for dilating blood vessels.
[0023]
(5) Effects on blood
  Artificial cultivated cordyceps alcohol extract has an inhibitory effect on thrombus formation in the abdominal aorta of rabbits. Wild cordyceps and artificially-grown cordyceps mycelium promote platelet formation, and the ultrastructure of the platelets formed is the same as normal.
[0024]
(6) Other clinical effects
  Cordyceps itself has an effect of improving liver function. HbsAg has a certain effect on seroconversion and can also significantly improve the plasma albumin of patients. Cordyceps has a remarkable inhibitory effect on the activity of monoamine oxidase (MAO-B) in the brain, and is beneficial for alleviating aging and maintaining the health of the elderly. In addition, Cordyceps has actions such as sedation, prevention of oxygen deficiency, anti-asthma, and anti-inflammation, and has actions similar to male hormones.
[0025]
(7) Toxicity
  When the cordyceps is injected into the abdominal cavity of a mouse, its LD₅₀The value is 21.7 ± 2.6 g / kg, and for intravenous injection, its LD₅₀The value is 24.5 ± 2.2 g / kg. Intravenous, intraperitoneal, or oral administration has proven to be less toxic.
[0026]
(8) Known components of Cordyceps and its activity
  Currently, components that can be isolated from Cordyceps include: nucleosides and nucleotides: adenine, adenosine, uracil, uridine, thymidine, ribosyl hypoxanthine, hypoxanthine, cordycepin (3'-deoxyadenosine), 3 '-L-lysylamino-3'-deoxyadenosine, N⁶-(2-Hydroxy) -adenosine. Alkaloid: opiocordin. Sugars: polysaccharides, D-mannitol. Sterols: ergosterol, ergosteryl-β-D-glucopyranoside, 22-dihydroergosteryl-β-D-glucopyranoside, 5α, 8α-epidioxy-5α-ergosta-6,22-dien-3β-ol. Fatty acids: palmitic acid, stearic acid, oleic acid, linoleic acid, and cholesterol palmitate. Natural products already isolated in cordyceps are adenine, adenosine, uracil, uridine, cholesterol palmitate, palmitic acid, and 5α, 8α-epidioxy-5α-ergosta-6,22-dien-3β-ol.
[0027]
[Problems to be solved by the invention]
  Cordyceps is a drug with a very wide pharmacological action and a very low toxicity, and its main action is immunomodulation and improvement of kidney function, so that systemic lupus erythematosus nephritis caused by immunity, especially clinical In addition, it has a considerable effect on type IV systemic lupus erythematosus nephritis, in which chronic mesangial proliferation and renal function are simultaneously degenerated. Previous studies have only used either Cordyceps complex or its crude product.
[0028]
[Means for Solving the Problems]
  In the present invention, research was conducted on the treatment and therapeutic effects of systemic lupus erythematosus nephritis using isolated natural products.
[0029]
  As a result, the present invention has found an active moiety isolated from Cordycops Sinensis (Berk) Sacc, a pharmaceutical composition of the active compound, and a method for improving kidney function using them. Furthermore, the present invention has also found a method for preventing and / or treating lupus erythematosus and / or IgA nephritis.
[0030]
  Accordingly, the present invention provides a pharmaceutical composition comprising active fraction F2, isolated from cordyceps, active compound H1-A, and optionally a pharmaceutically acceptable base.
[0031]
  In a preferred embodiment, the active fraction F2 is prepared by heating and drying the cordyceps angiosperm portion at 45-50 ° C., pulverizing it, immersing it in 20 times (w / v) methanol, and concentrating the extract under reduced pressure. Then, the crude extract is subjected to silica gel column chromatography, eluted with 1: 1 n-hexane / ethyl acetate mixed solvent, and the eluate is concentrated to obtain the extract.
[0032]
  In a more preferred embodiment, the active compoundIsolated from cordyceps (24R)- Ergosta -7,22- Jien -3b, 5a, 6b- Triol.
[0033]
  In a preferred embodiment, the pharmaceutical composition is for improving renal function, and in vitro, production of IL-2 by monocytes in peripheral blood activated by PHA and activated mesangial cells. Inhibits proliferation and, in vivo, inhibits lymph node proliferation in the MRL lpr / lpr animal model, inhibits the production of anti-ds-DNA autoantibodies, slows proteinuria progression, and prevents exacerbation of IgA nephritis be able to.
[0034]
  In a more preferred embodiment, it comprises a therapeutically effective amount of said F2 or H1-A for improving kidney function.
[0035]
  In another preferred embodiment, the pharmaceutical composition is for improving renal function for treating lupus erythematosus and / or IgA nephritis.
[0036]
  In a further preferred embodiment, the pharmaceutical composition comprises an effective amount of said F2 or H1-A for preventing and / or treating lupus erythematosus nephritis.
[0037]
  In a more preferred embodiment, the pharmaceutical composition comprises an effective amount of said F2 or H1-A for preventing and / or treating IgA nephritis.
[0038]
DETAILED DESCRIPTION OF THE INVENTION
  Hereinafter, the present invention will be further described with reference to the accompanying drawings.
[0039]
  The present invention provides a pharmaceutical composition for treating systemic lupus erythematosus nephritis and a method for treating systemic lupus erythematosus nephritis using the pharmaceutical composition, the pharmaceutical composition comprising Cordyceps Active fraction and active compound isolated from Sinensis (Berk) Sacc). Active fractions and active compounds isolated from Cordyceps Sinensis (Berk) Sacc have already been described in detail in Taiwan Patent No. 78584 by the inventor.
[0040]
  For the extraction and separation of the active ingredient of Cordyceps sinensis, the fruit body part of Cordyceps sinensis is taken, dried (45 to 50 ° C.), ground, immersed in 20 times volume methanol (w / v) and extracted. Further, the solvent is distilled off with a vacuum evaporator to obtain an extract. Then, the extract is subjected to silica gel column chromatography, and three kinds of solvents of hexane, ethyl acetate and methanol are mixed in various ratios, and are roughly divided into six fractions (F1 to F6) depending on the magnitude of polarity. For example, the separation procedure is shown in FIG. As a result of testing the biological activity, most of the activity is found in the eluate of F2 (hexane: ethyl acetate = 1: 1 (v / v)). Therefore, F2 is continuously separated, and silica gel column chromatography is again performed on F2, eluting with different polar solvents, and subdivided into 18 fractions (C1 to C18) according to polarity. As a result of testing the biological activity, most of the activity is found in the eluate of C11 (hexane: ethyl acetate = 1: 2 (v / v)). Therefore, separation is performed using preparative silica gel thin layer chromatography for C11. When the developing solution: hexane / ethyl acetate = 1: 1 (v / v) is developed twice, it is further subdivided into six fractions (T1 to T6).
[0041]
  As a result of testing the biological activity, the activity of T4 (Rf: 0.53 to 0.72) is relatively strong, and further, reverse phase high performance liquid chromatography (RP-HPLC) separation is performed on T4. The conditions used are as follows.
Column: Reversed phase type, 5C18 (for semi-preparation, 250 x 8mm)
Mobile phase: methanol
Detection: UV, wavelength 254nm
Flow rate: 2 ml / min
[0042]
  Using this HPLC method, five major fractions (H1-H5) were collected. As a result of measuring their activity, H1 (t_R: 10.0 to 10.9 minutes). Thereafter, the product is completely purified by another high performance liquid chromatography. The conditions used are as follows.
Column: reversed phase, cosmosil 5C18 (250 x 8 mm)
Protection column 10C 18 (50 x 4.6 mm)
Mobile phase: Methanol: H₂O = 90: 10 (v / v)
Detection: UV, wavelength 254nm
Flow rate: 2 ml / min
[0043]
  Finally, A and B compounds are obtained. According to the activity analysis, the main activity is the A component (hereinafter referred to as H1-A). The purity of H1-A is approximately 100% by HPLC purity analysis. The chemical structure of the purified H1-A compound was analyzed and analyzed by spectral methods such as mass spectrometry (MS) and nuclear magnetic resonance (NMR).Determine.
[0044]
  In the procedure for isolating Cordyceps natural products, the activity of each fraction is measured using the inhibitory action on mesangial cell proliferation activated by IL-1β and PDGFBB (platelet-derived growth factor) as an index. The method is as follows.
[0045]
  First, the mesangial cells to be cultured are trypsinized and washed three times with fetal bovine serum RPMI-1640 medium, and finally the cell suspension is washed with RPMI-1640 medium containing 2% fetal bovine serum. Incubate and adjust the concentration to 1 × 10 cells / ml. The mesangial cells adjusted to this concentration are placed in a 96-well culture dish (100 μl / well), and after 6 hours, 25 μl each of IL-1β (30 units / ml) and PDGF (500 units / ml) are added, Add 50 μl of various Cordyceps extract fractions (eg F1-F6, C1-C18, T1-T6, H1-H5). After 3 days of culture, “2-³Add 20 μl of “H” thymidine (1 μCi / well) and incubate for another 18 hours. Thereafter, the cell supernatant is removed, the cells are washed with PBS, and trypsin is added thereon to suspend the cells. With an automated cell collector, the cells are collected on glass fiber filter paper, the filter paper is dried, and the filter paper is placed in a counting vial. With a liquid scintillation counter, [2-³[H] thymidine is taken into mesangial cell DNA. Theoretically, if there are many cell divisions, “2-³H "thymidine is also increased. Therefore, the uptake rate is used as an index of cellular DNA synthesis. After treatment with various components, the radioactivity values (expressed in cpm) contained in the cellular DNA can be compared to determine whether this component has an inhibitory effect. Displays the amount of suppression as a percentage.
Percentage of inhibition (%) =
  (Group without medicine (cpm)-group with medicine (cpm)) / group without medicine (cpm) x 100
Percent inhibition:> 50% is recognized as having a significant inhibitory effect.
[0046]
  The separated active ingredient contains an active fraction and an active compound and is used in a pharmaceutical composition. These active fractions or active compounds are utilized alone or in the form of a pharmaceutical composition. One or more conventional excipients can be added to the compounds to make pharmaceutical compositions suitable for various uses. The active ingredients or active compounds according to the invention are made, for example, in tablets, capsules, granules, powders, liquids and other forms. Conventional excipients, binders, lubricants, colorants, disintegrants, and other materials can be used when making solid formulations for internal use.
[0047]
  Excipients include lactose, starch, talc, magnesium stearate, crystalline cellulose, methylcellulose, hydroxymethylcellulose, glycerin, sodium alginate, art (glue) and the like. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, art, shellac, sucrose and the like. Examples of the lubricant include talc, magnesium stearate, and the like. A colorant or disintegrating agent that is commonly used can be appropriately employed. The tablets can be coated by well-known methods.
[0048]
  Liquid formulations can be prepared in the usual ways to prepare these formulations in aqueous or oily suspensions, solutions, syrups or other forms. When preparing injections, pH adjusters, buffers, stabilizers, tonicity agents, local anesthetics, and others can be added to the compounds of the present invention. In addition, subcutaneous, intramuscular or intravenous injection preparations can be made in the usual manner. In making suppositories, the materials used as a substrate include, for example, oils, fats, cocoa butter, polyglycols and other similar substances.
[0049]
  The amount of the pharmaceutical composition prepared as described above varies depending on symptoms, patient weight and age, and the same amount is not always used. Generally, when used for an adult, the amount of the compound of the present invention may be about 0.01 to 2000 mg per day. This amount may be taken once daily, but can be taken 2 to 4 times a day.
[0050]
  Systemic lupus erythematosus is a chronic autoimmune disease as described above. The main cause of the pathology is that the various autoantibodies produced in the body cause the function of each system and organ of the body to be lost. Clinical manifestations are lymph node enlargement, vasculitis, and fatal systemic lupus erythematosus nephritis. MRL lpr / lpr mice are an animal model of spontaneous autoimmune disease. During the growth of mice, symptoms of human systemic lupus erythematosus occur, including the production of autoantibodies (anti-DNA antibodies), Includes arthritis, prominent lymphatic lesions and glomerulonephritis. Most mice lose progressive kidney function between 3 and 6 months. At 20 weeks, 50% of mice die. Kidney failure is a major cause of mouse death.
[0051]
  The pathological cause of MRL lpr / lpr lesions has not been elucidated at this time. Numerous references have reported research on the treatment of mice in a variety of treatment methods, including cyclophosphamide, cyclosporine, FK506, heparin, captopril, ornithine carboxylase inhibitors, neonatal thymectomy, homing These include monoclonal antibodies to the receptor, IL-2 / vaccinia recombinant vaccine, introduction of transgenic T cell genes, and the treatment of traditional Chinese medicine called immunoregulation. As described above, Cordyceps was reported as a Chinese medicine with immunomodulation, but there is also a literature reporting that it can increase the survival rate of systemic lupus erythematosus mice and suppress the production of anti-ds-DNA autoantibodies. is there. As in the examples described below, the present invention shows that in the results of in vitro tests of MRL lpr / lpr mice using isolated natural product H1-A, H1-A was activated by PHA (phytohemagglutinin). Effectively suppresses IL-2 production by blood monocytes.
[0052]
  In addition, in the following examples, F2 isolated from cordyceps can suppress the activation of renal stromal cells outside the body, and can also prevent the occurrence or worsening of IgA nephritis in the body. Indicates that F2 does not cause acute toxicity and alleviates the phenomenon of liver swelling induced by IgA IC. The active natural product (H1-A) purified from F2 has a good effect on an animal model that inhibits “occurrence of IgA nephritis in the body”.
[0053]
  Thus, the present invention further provides a method for treating lupus erythematosus and IgA nephritis. It also encompasses administering a therapeutically effective amount of a pharmaceutical composition of the invention to a patient desiring these treatment methods.
[0054]
  The present invention will be further described below with reference to examples, but the present invention is not limited to these examples. In the examples, when necessary, the results were expressed as mean values ± standard error, and statistical analysis was made by comparing differences between the two groups by Student's t-test.
[0055]
【Example】
Example 1: Effect of H1-A on IL-2 production by peripheral blood mononuclear cells (PBMNC)
  PBMNC are peripheral blood mononuclear cells isolated from the blood of normal and healthy blood donors. Cell concentration 2 × 10⁶Adjust to cells / ml. 2 × 10^FiveCells were placed in each well of a 96 well culture dish. Cells were cultured in RPMI-1640 culture medium with 2% fetal calf serum. Half of the cells were simultaneously treated with PHA (10 μg / ml) and different concentrations of H1-A (0 μmol / L, 3.125 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 9.375 μmol / L). The other half of the cells were treated with different concentrations of H1-A (0 μmol / L, 3.125 μmol / L, 12.5 μmol / L, 6.25 μmol / L, 9.375 μmol / L). After adding H1-A to the cells, 5% CO at 37 ° C.₂For 72 hours in an incubator. After 72 hours, cell supernatants were removed and analyzed. Using IL-2 ELISA Kit (T Cell Diagnosis Inc., Cambridge, MA), the concentration of IL-2 was measured, and the minimum concentration was 3.0 × 10^-11g / ml.
[0056]
  The effect of H1-A on activated or resting peripheral blood mononuclear cells,FIG.Shown in Resting peripheral blood mononuclear cells have low IL-2 production (262 ± 26.6 / ml), and H1-A has a significant effect on resting peripheral blood mononuclear cells producing IL-2. I didn't have it. PHA (10 μg / mL) stimulated IL-2 production of peripheral blood mononuclear cells, whereas H1-A suppressed IL-2 production. Its inhibitory action is related to the amount of drug. The statistical results show that there is a statistically significant difference in the inhibitory effect at the concentrations of 6.25 μmol / L and 12.5 μmol / L.
[0057]
Example 2: Treatment of MLR lpr / lpr mice with Cordyceps simple natural product H1-A
  Twelve MLR lpr / lpr mice were purchased from the Jackson Laboratories Animal Center. All mice were born to the same womb. These mice were all 12 weeks of age when they entered the study and all had symptoms of lymphadenopathy, hair loss, vasculitis and proteinuria. Before starting the experiment, another MLR lpr / lpr mouse born to the same womb was sacrificed and changes in renal pathology prior to the experiment were observed; the results already had highly proliferative glomerulonephritis The pathology resembled that of class IV systemic lupus erythematosus nephritis.
[0058]
  The mice were then randomly divided into 2 groups. In order to avoid the effects of body hormones due to gender, the sex ratio of the treatment group and the control group was 3: 3. Prior to the experiment, cordyceps (primitive complex containing H1-A) was first fed to another MLR lpr / lpr mouse. The result was that the Cordyceps complex could certainly improve systemic lupus erythematosus nephritis and was dose related. Its minimum effective concentration was 1% (wt / wt). Furthermore, the concentration of H1-A was calculated based on the extraction rate of H1-A extracted from cordyceps. The minimum effective amount corresponded to H1-A 40 μg / kg / day. Therefore, H1-A was placed in a minimal amount of alcohol and was administered to MLR lpr / lpr mice in the treatment group for 8 weeks. All mice were housed in a specific pathogen free (SPF) environment.
[0059]
(1) Effect of H1-A on survival period of MLR lpr / lpr mice
  The results of comparing the survival time of the treated and control mice are:FIG.It was as follows. All mice in the treatment group were alive until the end of the study. That is, although they survived to 20 weeks, two mice in the control group died of illness at 17 and 18 weeks. At the time of death, mice exhibited symptoms of systemic edema, severe proteinuria, and manure. Survival up to 20 weeks was 66.6% in the control group of mice compared to 100% in the treated group.
[0060]
(2) Effects of H1-A on lymphatic lesions and proteinuria in mice
  Lymph node size was measured and scored. The lymph node size is recorded every two weeks, and the score is calculated as follows. <5 mm is 0 point, 6 to 10 mm is 1 point, 11 to 15 mm is 2 points, 16 to 20 mm is 3 points, and 20 mm or more is 4 points. The score for each time was the sum of the scores for the armpits and radial lymph nodes, and was used as an index of the size of the lymph nodes at the time of recording.
[0061]
  Differences in lymph lesion processes between treated and control mice were compared. Retrograde changes may occur in lymphatic lesions 6 weeks after receiving H1-A treatment, but a statistically significant inhibitory effect was observed after 8 weeks from the start of treatment with H1-A mice. (FIG.checking).
[0062]
  Urine fluid was obtained by massaging the bladder because the urine of MLR lpr / lpr mice was low and difficult to obtain. Urine fluid was collected every 2 weeks. The collected urine fluid was stored in a −70 ° C. freezer until analysis. For urine protein, the ratio of albumin (μg / ml) and creatinine (mg / kg) in urine was recorded. For determination of albumin in urine, an ELISA kit of albumin diagnostic reagent (Sigma 631-2; Sigma Chemical Co., ST. Louis, MO) was used. The standard is purified mouse albumin (Cappel, Organon Teknika, Durham, NC) diluted at different concentrations, with a concentration range of 5-50 μg / ml.
[0063]
  Regarding the effect of H1-A on mouse proteinuria, mice in the control group have progressive proteinuria, but in the treatment group, the urinary albumin / creatinine values decrease sequentially after 4 weeks of H1-A treatment. There was a trend. The numerical difference between the two groups was statistically significant when the mice were 20 weeks old. In the urine fluids of the control group and the treatment group, the ratio of albumin “μg / ml” to creatinine “mg / kg” was 0.88 ± 2.94 and 0.293 ± 0.19, respectively (P <0.05) (FIG.checking).
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(3) Effects of H1-A on kidney function in MLR lpr / lpr mice
  In order to examine whether H1-A can improve the kidney function of the mouse and conversely, it becomes nephrotoxicity, the blood of the MLR lpr / lpr mouse is taken every 2 weeks by taking blood from the retro-orbit. I got blood. Furthermore, serum was obtained by centrifugation at 1000 rpm for 5 minutes. All obtained sera were stored in a -70 ° C freezer until testing. Sera from various stages of mice were obtained and blood urea nitrogen and creatinine measurements were taken. Serum creatinine was measured colorimetrically with picric acid (Sigma 555A). The measurement of urea nitrogen in the blood was performed with the Urease Assay Kit (Sigma 640-A). All are the results of a double test. Displayed as mean ± standard error and mg / dl.
[0065]
  MRL lpr / lpr mice in the control group gradually deteriorated in kidney function during the follow-up of the test, and the blood urea nitrogen level increased from 25 ± 18 mg / kg to 35.94 ± 3.75 mg / kg. The value of creatinine increased from 1.33 ± 0.09 mg / kg to 3.04 ± 0.47 mg / kg. In relative terms, the kidney function of the treatment group is better than the control group, and the urea nitrogen and creatinine values in the blood of the mice are 24 ± 1.8 mg / kg and 1.23 ±, respectively, when the mice are 20 weeks old. It was 0.19 mg / kg. At 20 weeks, the two groups had statistically significant differences in blood urea nitrogen and creatinine levels (P <0.05) (FIG.checking).
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(4) Effect of H1-A on the production of anti-ds-DNA autoantibodies by MLR lpr / lpr mice
  Sera from 12, 16 and 20 week old mice were collected for analysis of anti-ds-DNA autoantibodies. Analysis of anti-ds-DNA IgG group autoantibodies in serum was performed by ELISA. First, a 96-well plate (Corning 25801 ELISA Plate) was covered with 100 μl of methylated bovine serum albumin (10 μg / ml). After standing at 4 ° C. overnight, the plate was washed 3 times with PBS containing 0.05% Tween 20. Then, 100 μl of fetal bovine thymus DNA (concentration: 2.5 μg / ml) containing 0.1 mmol / l phosphate was added to each well, left overnight at 4 ° C., and further three times with PBS containing 0.05% Tween 20 After washing, it was dried. Subsequently, each well was blocked with 200 μl of 1 mg / ml gelatin, and after 2 hours at 37 ° C., it was washed with PBS containing 0.05% Tween 20 three times. Separately, a mouse serum sample prepared in advance was diluted 1: 500, 100 μl was added per well, and washed after 1 hour. On top of this, 100 μl of goat anti-mouse IgG (H + L) -horseradish peroxidase (1: 2000 in PBS) was added, washed after 1 hour at 37 ° C. and finally 2,2′-azino-di- [ 100 μl of a base solution containing 3-ethylbenzthiazoline sulfonate (6)] and hydrogen peroxide was added. After 30 minutes at room temperature, 50 μl of 10% sodium dodecyl sulfate was added to stop the reaction. The absorbance was measured at 405 nm, and the result was expressed as an average value ± standard error.
[0067]
  The results comparing the differences in IgG and anti-ds-DNA autoantibodies in mouse sera from treated and control groups at 12 weeks, 16 weeks and 20 weeks were:FIG.It was as shown in. That is, the anti-ds-DNA value in the mouse blood of the control group increased from 0.141 ± 0.036 to 0.198 ± 0.047 after 8 weeks of follow-up, while the absorbance at 405 nm of the treatment group was 0.172 ± 0.009. Dropped to 0.112 ± 0.015. After 8 weeks of treatment with H1-A, that is, when the mice are 20 weeks old, the anti-ds-DNA autoantibody values in the blood of the mice in the treatment group and the control group are statistically significant. (P <0.005) (FIG.checking).
[0068]
(5) Effect of H1-A on changes in renal histopathology of MRL lpr / lpr mice
  Except for two mice that died at 17 and 18 weeks, all mice were sacrificed after 20 weeks by cutting the cervical spine. Mouse kidney tissue (including spontaneously dead mice at 17 and 18 weeks) was removed. Half of the kidney tissue was snap frozen and the other half of the kidney tissue was fixed with Carson-Milloning's solution and prepared for sectioning and electron microscopy. From the rapidly frozen tissue, a slice having a thickness of 4 μm was cut out by a cryosectioning machine and prepared for examination with a fluorescence microscope.
[0069]
  The fixed kidney tissue is first dehydrated with different concentrations of alcohol, and then a paraffin-embedded 3 μm section is cut out and then stained with periodic acid-Schiff reagent and prepared for optical microscopy. did. Semi-quantitative analysis was performed by the method of Gesualdo et al. Briefly, at least 40 kidney glomeruli were examined per piece of glass on which the renal cortex was placed, and 0-3 depending on the degree of mesangial cell proliferation.⁺The various grades were counted. 0 indicates normal; trace amounts indicate that 2-3 nuclei are found in the mesangial part; 1⁺Indicates light growth in mesangial cells and 4-6 nuclei in each mesangial part; 2⁺Indicates moderate growth in mesangial cells, with 4-6 nuclei present in most mesangial parts and 7 or more nuclei in other parts; 3⁺Indicates significant proliferation in mesangial cells, with 7 or more nuclei present in each mesangial portion. Two judges (L.F.S. and W.H.L) judged their own pathological sections, and the entire study was designed in a double-blind manner. The total number of cell proliferation counts for each sample was calculated in the following manner. Total proliferation index = (percentage when the glomerular proliferation index is 0 × 0) + (percentage when the glomerular proliferation index is very small × 0.5) + (the glomerular proliferation index is 1)⁺(Percentage when x1) + (The glomerular proliferation index is 2)⁺(Percentage when x2) + (The glomerular proliferation index is 3)⁺The percentage of when x3). The total proliferation index for each sample was 0-300.
[0070]
  For examination with a fluorescence microscope, the frozen sections were first air-dried, fixed with acetone for 10 minutes at room temperature, and then washed with PBS. Each section of each sample was stained with fluorescein isothiocyanate-conjugated goat anti-mouse IgA, IgG, IgM and C3 (Cappel) and again observed with a fluorescence microscope (Olympus). The judgment method was based on the counting method described above. In simple terms, the observer examines more than 40 renal glomeruli and 0-3 for each glomerulus.⁺A grade count was made (0 indicates no fluorescence; trace indicates fluorescence but not clearly visible; 1⁺Shows mild fluorescence; 2⁺Indicates moderate fluorescence staining; 3⁺Indicates strong fluorescent staining). The total fluorescence intensity index was calculated by the following method. Total fluorescence intensity index = (renal glomerular percentage when fluorescence intensity is 0 × 0) + (renal glomerular percentage when fluorescence intensity is very small × 0.5) + (fluorescence intensity is 1)⁺Kidney glomerular percentage x1) + (fluorescence intensity is 2)⁺Kidney glomerular percentage x2) + (fluorescence intensity is 3)⁺The percentage of kidney glomerulus when x is 3). The total fluorescence intensity index for each sample was 0-300.
[0071]
  Severe kidney growth was observed in the kidney tissue sections of the control group mice in both the two mice that died spontaneously and the mice sacrificed at 20 weeks. In the treatment group, significant histopathological changes were seen after receiving H1-A treatment (FIG.(See A and B). Mesangial proliferation was clearly suppressed, and there were also retrograde changes. The kidney sections of the treatment group were stained with hematoxylin-eosin and all showed mild mesangial growth. There were no significant differences between the treatment group and the control group with respect to renal tubules, renal stroma, or vascular changes. Also, no statistical difference was seen between the two groups with fluorescent staining (see Table 1).
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[Table 1]

[0073]
  From the above results, pure natural product H1-A isolated from Cordyceps sinensis was able to suppress IL-2 production in vitro more than peripheral blood mononuclear cells activated by PHA. In the MRL lpr / lpr animal model, it was also possible to inhibit lymph node proliferation and suppress the production of anti-ds-DNA autoantibodies. It was also possible to extend the survival time of MRL lpr / lpr mice. At the same time, mesangial proliferation of the kidney glomeruli was suppressed and the progression of proteinuria was delayed. Surprisingly, no significant change was observed in the deposition of immune complexes in this study. The results of this study revealed that this isolated natural product with a well-defined chemical structure has the potential to treat autoimmune diseases in MRL lpr / lpr mice. In addition, there is no relation between serum anti-ds-DNA autoantibody concentration, renal histopathological changes and kidney immune complex deposition, but in the future, for the cause of systemic lupus erythematosus nephritis, It becomes possible to provide more examination space.
[0074]
  The in vitro test results of the present invention showed that H1-A can effectively suppress the production of IL-2 by peripheral blood monocytic cells activated by PHA. The effect of this drug is similar to cyclosporin A, a well-known immunosuppressant. As described above, H1-A, which is a primitive complex of Cordyceps sinensis, has an immunosuppressive action on cellular immunity. This result is similar to that in cyclosporin A skin transplantation. In other words, H1-A is more appropriate to be called an immunomodulator. Cyclosporine A is said to be effective in treating human systemic lupus erythematosus. Cyclosporine A is selectively used for T lymphocytes. Therefore, it is used for treatment of lesions mainly composed of T cell responses. Cyclosporine A also has a suppressive action on reactions associated with several antibodies, but its main action is on the reaction produced by T helper cells. In comparison, cyclosporin A has little effect on the antibody response generated when the antigen directly stimulates B cells. H1-A may have a mechanism of action similar to that of cyclosporin A, since the inhibitory action against anti-ds-DNA antibodies is noticeable. This is because the production of anti-ds-DNA is a process of T cell regulation by antigen activation. H1-A also has an inhibitory effect on lymph node proliferation whose main symptom is T cell proliferation. The new perspective on the mechanism of systemic lupus erythematosus is CD4⁺T cells are the main cause of MRL lpr / lpr mouse autoimmune disease development. Thus, currently the cause of such illness is due to the imbalance of T cell function or lack of cooperation between T cells and B cells. Anti-CD3, anti-CD4, and anti-CD8 monoclonal antibodies have been reported to be effective in the treatment of murine lupus erythematosus. Therefore, it is speculated that the H1-A therapeutic effect is exhibited by suppressing the production of T cells or suppressing the production of Th1 / Th2.
[0075]
  Due to the inhibitory effect on Th1 and Th2, the production of anti-ds-DNA antibodies in the mouse body was significantly reduced. This may be due to the suppression of kidney local Th1 production. This stopped the proliferation of mesangial cells in the glomeruli. It is necessary to further prove these hypotheses in future studies. The present invention provided three possible interpretations of the absence of changes in immune complex deposition in kidney sections.
[0076]
  First, the unrelatedness between renal histopathological changes and immune complex deposition is common clinically. Therefore, it is impossible to interpret all the phenomena of systemic lupus erythematosus nephritis with immune complexes. Other intervening factors include cellular factors or chemical factors. Therefore, the inhibitory action of H1-A on mesangial proliferation may be exerted through a mechanism other than lowering immune complex deposition.
[0077]
  Secondly, removal of already deposited immune complexes takes a lot of time. According to clinical experience with type IV systemic lupus erythematosus nephritis, after patients receive treatment with cyclophosphamide, the anti-ds-DNA antibody level in the blood decreases first as the symptoms of the disease alleviate. The elimination of immune complexes in kidney tissue is slower than clinical symptom improvement. Usually, after at least 6 months of treatment, significant changes in kidney tissue are first seen.
[0078]
  Third, H1-A does not improve the ability of macrophages to eliminate immune complexes. Thus, H1-A is a potential isolated natural product for treating human systemic lupus erythematosus. H1-A is safer than cyclosporin A. Cordyceps is used in traditional Chinese medicine because it has not been reported to date on renal toxicity, despite having a history of over a thousand years. Conversely, Cordyceps has been reported to be able to effectively avoid nephrotoxicity caused by cyclosporin A and aminoglycosides. This study also proved this viewpoint. Kidney slices do not show any nephrotoxicity, and conversely, H1-A has even a protective effect on the renal function of MRL lpr / lpr mice.
[0079]
  From the viewpoint of immune mechanisms, many literatures have recently described that T cell function in patients with systemic lupus erythematosus is defective. It includes that IL-2 production is reduced and that the response of IL-2 induced T cell proliferation is also significantly reduced. Gutierrez-Ramos et al. Successfully treated MRL lpr / lpr mice with an IL-2 / vaccinia recombinant vaccine. Some so-called immunomodulators have been described to correct defects due to IL-2 and promote cell division and peripheral T cell activity in the thymus. However, one person gave a completely opposite opinion. Cuadrado et al. Reported a relatively normal and significant increase in IL-4, IL-2, and sIL-2R in the blood of patients with systemic lupus erythematosus. In contrast, Raz et al. Reported that the introduction of IL-2 cDNA into the body of MRL lpr / lpr mice exacerbated the pathology and further promoted autoantibody production. The results of the inventors are almost consistent with the latter opinion. In vitro, H1-A suppresses IL-2 production in peripheral mononuclear cells; in the MRL lpr / lpr mouse animal model, H1-A improves clinical symptoms and renal tissue pathology Can do.
[0080]
  As described above, the present invention uses the isolated natural product H1-A having a well-defined structure isolated from Chinese herbal medicine, Chinese herbal medicine, MRL lpr / lpr of autoimmune disease model. Succeeded in improving the treatment effect in the body.
[0081]
Example 3: Effect of Cordyceps extract (1% and 0.5% F2) on mice with induced IgA nephritis
[0082]
1. Methods for inducing IgA nephritis: The antigen used is R36A and the antibody is a-R36A-IgA-mAb. First, 2 mg of antibody was injected into the abdomen of Balb / c mice, and 2 hours later, 100 μg of antigen was injected into the tail vein. Then, every 24 hours, 100 μg of antigen was injected (continuous for 1 week), which caused immune complexes to deposit in the renal interstitium, and hematuria and proteinuria appeared about 5 days after the first injection.
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2. Mice that induced IgA nephritis were divided into a dosing group (n = 6) and a control group (n = 6). The dosing group received 1% and 0.5% F2. Each day, each mouse received about 3 g of normal diet. The mice in the test group and the control group were measured daily for hematuria and proteinuria, and after 7 days, the mice were sacrificed. The kidneys were removed for histopathology and immunofluorescence and observed for changes in serum ALT, AST and liver cholesterol, cytochrome P450.
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I. Measurement of hematuria and proteinuria:
The test was performed with a urine test paper (Urine Strip).
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II. Renal histopathology examination:
  The kidney tissue was fixed with 10% neutral formalin, dehydrated with increasing concentrations of alcohol, embedded in paraffin, cut into 4 μm thick sections, stained with hematoxylin-eosin, and observed with an optical microscope. 50 glomeruli are observed for each mouse, and the degree of glomerular destruction is divided into 5 grades. The higher the score, the higher the degree of glomerular destruction. Finally, the points are accumulated and displayed as the total number (Total).
[0086]
III. Immunofluorescence test:
  Kidney tissue was embedded with O.C.T.compound and stored in a -70 ° C freezer. In performing immunostaining, 4 μm tissue sections were cut with a cryosection machine and fixed with acetone, followed by direct immunofluorescence staining with fluorescently labeled goat anti-mouse IgA, goat anti-mouse C3, and goat anti-fibrinogen. And observed with a fluorescence microscope. The fluorescence staining intensity is divided into four grades, and a higher score indicates stronger fluorescence intensity, and higher IgA, IC deposition and C3 complement activation.
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3. Statistical method:
  Hematuria, proteinuria, histopathology and immunofluorescence were analyzed by chi-square test. Statistics of serum ALT, AST and liver cholesterol, cytochrome P450 were analyzed by Student's t-test. P <0.05 is considered statistically significant.
[0088]
result:
1. Hematuria and proteinuria are shown in Table 2.
[0089]
[Table 2]

[0090]
  Five days after the antigen injection, hematuria and proteinuria appeared. On day 7, significant reductions in hematuria and proteinuria were seen in the 1% F2 and 0.5% F2 groups (P <0.05).
[0091]
2. The results of renal histopathology examination are shown in Table 3.
[0092]
[Table 3]

[0093]
  In terms of the total number, the 1% F2 group significantly reduced the degree of destruction of the glomeruli (P <0.05), but the 0.5% F2 group showed no statistically significant difference.
[0094]
3. The results of the immunofluorescence test are shown in Tables 4 and 5.
[0095]
[Table 4]

[0096]
[Table 5]

[0097]
  From the Total evaluation, both the 1% F2 and 0.5% F2 groups of medication had reduced IgA IC deposition or C3 complement activation in proportion to the drug dose compared to the control group. . The 1% F2 group showed a statistically significant difference compared to the control group (P <0.05).
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4). The results of the liver function test are shown in Table 6.
[0099]
[Table 6]

[0100]
  There was no difference between the dosing group and the control group in terms of liver weight and body weight: from the 100-fold ratio of liver weight to body weight, the 1% F2 group was 6.75 ± 0.09 and the control group was 7.12 ± 0.12. Met. 1% F2 clearly reduced liver weight (P <0.05). Serum ALT and AST did not change significantly between the dosing group and the control group. The cholesterol content in the liver was not statistically significant between the dosing group and the control group. The content of cytochrome P450 in the liver was 0.31 ± 0.03 nmol / mg protein in the 1% F2 group and 0.23 ± 0.01 nmol / mg protein in the control group. 1% F2 significantly increased the cytochrome P450 content (P <0.05). In terms of the ratio of liver weight to body weight, the acute toxicity test showed liver swelling when 2% F2 was administered. In an animal model in which IgA nephritis was induced, liver atrophy was observed when 1% F2 was administered. These show the opposite results. In the acute toxicity test, normal ICR mice were used as a control group, and the ratio of 100 times liver weight to body weight was 5.44 ± 0.17. In the Balb / c mouse test in which IgA nephritis was induced, normal feeding was used as a control group. An animal in which IgA nephritis has been induced in advance has a ratio of 100 times liver weight to body weight of 7.12 ± 0.12, and a mouse swelling phenomenon is observed in mice with IgA nephritis. The animals used for the two studies were of different breeds. Furthermore, when normal diets were raised in normal Balb / c mice using the method of “bred with Cordyceps extract”, the ratio of liver weight to body weight 100 times was 5.50 ± 0.24.FIG.This was consistent with the normal 100-fold liver weight to body weight ratio (5.44 ± 0.17) in ICR mice. This showed that IgA IC itself causes a significant liver swelling phenomenon (P <0.05). The mechanism is unknown. However, administration of 1% F2 significantly reduced IgA IC-induced liver swelling (P <0.05) but failed to recover to normal values.
[0101]
Example 4: Safety to H1-A cells in vitro
  We examined the activity and toxicity of H1-A to cells,FIG.It was shown to. TopmostFIG.(A) is the result of measuring the activity of cells by trypan blue staining. Three groups cultured for 72 hours (only human mesangial cells, human mesangial cells + IL-1 and IL-6, and human mesangial cells + IL-1) It was shown that there is no difference in the cellular activity of IL-6 and 40 μM / ml H1-A). Since dead cells release LDH, in the same test (a), after 72 hours, the concentration of LDH in the culture was measured, and the result wasFIG.Shown in (b). There was no statistical difference between the three groups. If the cells survive, mesangial cells also grow. Therefore, the state of cell proliferation was analyzed by the MTT method. The resultFIG.Shown in (c). It can be seen that there was no statistical difference in the three groups.
[0102]
Example 5: Effect of H1-A on mice induced with IgA nephritis
  The IgA nephritis mice induced in Example 3 were injected with 10 mg / ml H1-A once in the abdomen and continuously for 7 days.
[0103]
  The effects of IgA nephritis mice in the treatment group and hematuria / proteinuria in the control group were compared. From the results shown in Table 7, it can be seen that the hematuria / proteinuria of the treatment group is significantly improved and statistically significant.
[0104]
  Regarding the renal tissue pathological change, severe mesangial growth and mesangial cell proliferation were observed in the control group, but the treatment group showed a marked improvement in histopathological change after receiving treatment with H1-A. . The kidney sections of the treatment group showed only slight mesangial growth with hematoxylin-eosin staining, and the improvement in fluorescence staining is shown in Table 7. The treatment group has a significant improvement and is statistically significant.
[0105]
[Table 7]

[0106]
【The invention's effect】
  F2 isolated from Cordyceps can inhibit the activation of renal stromal cells outside the body and can prevent the occurrence or worsening of IgA nephritis in the body. F2 has little acute toxicity and can also reduce liver swelling induced by IgA IC. The active natural product (H1-A) isolated from F2 is an animal model that prevents "internal IgA nephritis" and has a good therapeutic effect.
[0107]
  Although the present invention has been described with reference to preferred embodiments, it is to be understood that various modifications thereof will be apparent to those skilled in the art upon reading this specification. Accordingly, it is to be understood that the invention disclosed herein is intended to include these modifications as fall within the scope of the appended claims.
[Brief description of the drawings]
FIG. 1 is a flowchart showing a process of separating an active fraction and an active compound from cordyceps.
[FIG.This is a graph showing the effect of H1-A of the present invention on the production of IL-2 by peripheral blood mononuclear cells. ●: H1-A only; ▽: Stimulated mononuclear cells with PHA (10 μg / ml) + H1-A.
[FIG.The graph shows the effect of H1-A on the survival time of MRL lpr / lpr mice. ■: Treatment group administered with H1-A; ○: Untreated control group.
[FIG.The graph shows the effect of H1-A of the present invention on lymphatic lesions in MRL lpr / lpr mice. ■: Treatment group administered with H1-A; ○: Untreated control group.
[FIG.The graph shows the effect of H1-A of the present invention on proteinuria in MRL lpr / lpr mice. ■: Treatment group administered with H1-A; ○: Untreated control group.
[FIG.The graph shows the effect of H1-A of the present invention on kidney function in MRL lpr / lpr mice. ■: Treatment group administered with H1-A; ○: Untreated control group.
[FIG.The graph shows the effect of H1-A of the present invention on the production of anti-ds-DNA autoantibodies in MRL lpr / lpr mice. ■: Treatment group administered with H1-A; ○: Untreated control group.
[FIG.The graph shows the effect of H1-A of the present invention on cell activity. ■: control group, human mesangial cells only; □: human mesangial cells + IL-1, IL-6; □: human mesangial cells + IL-1, IL-6 and H1-A 40 μM / ml for 72 hours culture. (A) Observation of cell activity by trypan blue staining; (b) Observation of cell death by measurement of released LDH; (c) Observation of cell proliferation by MTT test.

Claims (1)

(24R)- Ergosta -7,22- Jien -3b, 5a, 6b- A pharmaceutical composition for preventing and / or treating lupus erythematosus nephritis, comprising triol as an active compound.

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